CN103930446A

CN103930446A - Structures surface-coated with pseudomonas pilin peptide

Info

Publication number: CN103930446A
Application number: CN201280029572.1A
Authority: CN
Inventors: R.T.欧文; E.M.戴维斯; D.李; D.A.马鲁夫
Original assignee: Arch Biophysics Inc
Current assignee: Arch Biophysics Inc
Priority date: 2011-04-14
Filing date: 2012-04-13
Publication date: 2014-07-16
Also published as: US20140220368A1; US9422450B2; WO2012139208A1; US20110293684A1; EP2697261A4; CA2833041A1; US9096775B2; EP2697261A1; AU2012243395A1; JP2014517817A

Abstract

The present invention relates to a methods and composition in which a D-form or retro-inverso Pseudomonas pilin peptide, optionally having a polyethylene glycol moiety covalently attached to the peptide, is bound to a substrate. The disclosed methods and composition are useful for reducing the frictional coefficient, inhibiting biofilm formation by non-Pseudomonas bacteria, for reducing the inflammatory response to a material, for reducing corrosion of metals, and in biosensor applications.

Description

The structure of surface coated pseudomonas pilin peptide

Invention field

The present invention relates to adopt D type or contrary-inversion (retro-inverso) IV type Pseudomonas aeruginosa ( p. aeruginosa) (T4P) field of the material coating of pilin peptide, described pilin peptide is optionally conjugated to polyethylene part; And relate to its methods and applications.

background

The IV type pili of bacterium is that troop (colonization) and virulence (for many Gram-negative bacterias) of host is necessary, and may in the pathogeny of some gram-positive microorganisms, work.IV type pili stretches out and mediates and the specificity on biological and abiotic surface is adhered to from bacterium surface.Be responsible for the pili binding domains of this combination at 12-17 Er Liuhuan district (disulfide-loop) coding that is being positioned at the C-end region of this albumen, and only contain the two sulphur cyclic peptide that the synthetic peptide in this district is for example comprised of the residue 128-144 from Pseudomonas aeruginosa IV type pilin, shown and biology and abiotic surface bonding.

The inventor and colleagues are verified recently, the derivative protein nano pipe (PNT) of pilin is attached to stainless steel with high-affinity, and it is (tip-associated) of the combination of C-end end that this binding events demonstrates, by the synthetic peptide competitive inhibition PNT combination corresponding to IV type pilin peptide binding domains, carry out (Yu, B. wait people, J. Bionanoscience, 1:73-83 (2007).The inventor and colleagues further prove subsequently, pilin peptide derived from C-end receptors bind structural domain, for example, when being attached to abiotic surface (stainless steel, tin, aluminium, titanium, chromium, plastics, glass, silicate, pottery and composition thereof), can suppress the formation (U.S. 20080287367) of bacterial biof iotalm on the surface of this coating.

Recently, the inventor finds, contain some pure surface properties that the synthesis bacterium hairless protein peptide of two sulphur rings and the combination of some metals derived from IV type Pseudomonas aeruginosa (T4P) pilin C-end receptor binding protein significantly strengthen metal,, be independent of the character of biofilm formation, and in some metals, change surperficial electronic property, make in a certain way it can be used for (USSN 12/899,958) in biological example sensor application.

summary of the invention

On the one hand, the present invention includes and process pipeline to reduce the method for the frictional drag of the fluid that flows through this pipeline, by introduce the step of the liquid that carries conjugate to described pipeline, described conjugate is following conjugate: (i) contain derived from two sulphur rings of the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin and contain and be positioned at the N-end side of described ring or the 0-10 of C-end side or this two end side extra D type of residue or the synthesis bacterium hairless protein peptide of contrary-inversion and (ii) polyalkylene glycol moiety, the amount of described conjugate is effectively by making pilin peptide be attached to the frictional drag that duct wall reduces the fluid that flows through pipeline.

Described pilin peptide conjugate can be the D type pilin peptide with the polyalkylene glycol moiety that is covalently attached to the C-end of described peptide or the one or both ends of N-end.Polyalkylene glycol moiety in described peptide conjugate can have the molecular weight between 0.2-500 kDal.Described pilin peptide can have the sequence that is defined as SEQ ID NO:10, comprises and is defined as SEQ ID NO:3,4 or 9 exemplary sequence.

The accompanying inner surface of pipeline of described pilin peptide conjugate can be metal, polymkeric substance, pottery or silicate surfaces.Exemplary metal comprises stainless steel, carbon steel, titanium, copper, brass, tin, iron, silver and magnesium and alloy thereof.

The amount or the surface density that are attached to the pilin peptide conjugate of inner-walls of duct can be enough to reduce for example stainless erosion rate of corrodible metal, and/or are enough to reduce the frictional drag of fluid, and described fluid is for example introduced ducted containing particle loaded fluid.

Can introduce in ducted fluid by the normal operation period described peptide conjugate regularly being joined, pilin peptide conjugate is introduced in pipeline.

On the other hand, the present invention includes the metal having with one or more exposures of conjugate coating, polymkeric substance, the goods of pottery or silicate surfaces, described conjugate is following conjugate: (i) contain derived from two sulphur rings of the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin and contain and be positioned at the N-end side of described ring or the 0-10 of C-end side or this two end side extra D type of residue or the synthesis bacterium hairless protein peptide of contrary-inversion and (ii) polyalkylene glycol moiety, the amount of described conjugate is enough to reduce the frictional coefficient of described one or more exposed surfaces.Exemplary pilin peptide as mentioned above.

One or more exposed surfaces of described goods can be metallic surfaces, the surface of stainless steel, carbon steel, titanium, copper, brass, tin, iron, silver and magnesium and alloy thereof for example, and the peptide conjugate that is coated with described surface can be enough to reduce the erosion rate on described surface, and/or the amount that is enough to reduce the frictional drag of the fluid flow through described pipeline exists.

In one embodiment, described goods are that width dimensions is 100 microns or less microfluidic channel, and its inner wall surface is with peptide conjugate coating and be coated with this surperficial peptide conjugate and exist with the amount that is enough to reduce fluid and flows through the frictional drag of described passage.

In the 2nd embodiment, described goods comprise sand particle, and the frictional drag of pipeline is flow through in enough described peptide conjugate coating for its outside surface to reduce the particulate slurry of this coating.

In the 3rd embodiment, described goods are the machines with movable part, and surface of its coating peptide is moving contact each other.

In the 4th embodiment, described goods are electron device or the elements with the metallic surface of the exposure being coated with described peptide conjugate.

In the 5th embodiment, described goods are the orthopedic implant devices with stainless steel or titanium surface, and the described peptide conjugate that is coated with this surface exists to be enough to suppress the amount of biofilm formation on this coating surface.

Again on the one hand, the present invention includes a kind of goods, described goods comprise that (i) has the surperficial element of exposure, described surface can be used as the substrate of biofilm formation under common working conditions by non-pseudomonas bacterium, (ii) with the D type of described surface bonding or the synthesis bacterium hairless protein peptide of contrary-inversion, the two sulphur rings that described synthesis bacterium hairless protein peptide contains derived from the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin are positioned at the N-end side of described ring or the 0-10 of C-end side or this two end side extra residue with containing, optionally be covalently attached to the polyalkylene glycol moiety of described peptide, the amount of described peptide suppresses the biofilm formation of the non-pseudomonas bacterium of this class effectively.Exemplary repressed bacterium comprise Listera ( listeria) or staphylococcus ( staphylococcus).

Again on the one hand, present invention resides in the method for the surperficial metallic substance of processing the grain boundary district with exposure in order to reduce the improvement of the erosion rate of described material.Described improvement comprises: the Surface Edge battery limit (BL) of the exposure in described material and the synthesis bacterium hairless protein peptide of D type or contrary-inversion are contacted, the two sulphur rings that described synthesis bacterium hairless protein peptide contains derived from the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin are positioned at the N-end side of described ring or the 0-10 of C-end side or this two end side extra residue with containing, optionally be covalently attached to the polyalkylene glycol moiety of described peptide, the amount of described peptide suppresses the erosion rate of described material effectively.Exemplary metal comprises stainless steel, carbon steel, titanium, copper, brass, tin, iron, silver and magnesium and alloy thereof.Exemplary pilin peptide and pilin peptide conjugate are as mentioned above.

Be attached to that electronics work content that the peptide of described metal or the amount of peptide conjugate can effectively make the grain boundary district that exposes is changed to few 0.3eV EFW unit and the hardness increase at least 20% in the grain boundary district that makes to expose, described hardness is measured with the nano impress of given force impact (striking) metallic surface generation by the tip with atomic force microscope.When metal is stainless steel, in conjunction with peptide or the amount of the peptide conjugate of combination can effectively make the erosion rate of coating surface reduce at least about 30%, described erosion rate is by measuring through surperficial corrosion current.

When metallic substance is porous or when netted, contact procedure can be implemented so that the definite internal surface of pilin peptide and hole by described material or mesh is combined.When metallic substance has the grain boundary district of exposure, combination that contact procedure can effectively make pilin peptide and described grain boundary regioselectivity, the surface by place, priority protection Qi grain boundary district strengthens hardness and the corrosion resistance of metallic surface thus.

Also disclosed is to suppress being designed to implant the method for the Inflammatory response of the medical device in experimenter.Described method comprises: before implanting described device, with synthesis bacterium hairless protein peptide, be coated with the surface of the exposure of described device, described synthesis bacterium hairless protein peptide contains (i) derived from two sulphur rings of the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin, (ii) be positioned at the N-end side of described ring or the 0-10 of C-end side or these two end sides extra residue, (iii) the D-amino acid by contrary-inversion (RI) type forms, and optionally has the polyalkylene glycol moiety being covalently attached on described peptide.Preferred peptide and peptide conjugate are as mentioned above.

On the other hand, the present invention includes the biosensor device for detection of analyte.Described device comprises: (a) conducting metal substrate, it has D type (RI) the synthesis bacterium hairless protein peptide that (i) biosensor surface (ii) is combined with described substrate surface, the two sulphur rings that described synthesis bacterium hairless protein peptide contains derived from the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin are positioned at the N-end side of described ring or the 0-10 of C-end side or this two end side extra residue with containing, optionally there is the polyalkylene glycol moiety that is covalently attached to described peptide, (iii) be covalently attached to directly or indirectly the acceptor of described pilin peptide, (b) for detection of the detector of the change in electrical properties through substrate surface of the combination of the acceptor of response analysis thing associated ligands and surface bonding.

Described biosensor substrate can be formed by stainless steel, and pilin peptide combined thereon can be D type pilin peptide, is optionally conjugated to polyalkylene glycol moiety.In one embodiment, can use the combination of pilin peptide, for example, use PEG-D-pilin peptide conjugate with the non-specific interaction of minimizing and sample compound, with RI-pilin peptide (being with or without peg moiety) so that target ligands is connected to biosensor surface, or directly by the covalently bound of part and peptide or by indirect mode, as described below.

Described acceptor can be by coiled coil E/K spiral to being covalently attached to pilin peptide, and one of described spiral centering is covalently attached to pilin peptide, and another is connected on acceptor.

Again on the one hand, the present invention includes compound is covalently attached to by stainless steel, tin, the method of one or more exposed surfaces of the substrate that iron or titanium form, described method is passed through following steps: (a) make one or more exposed surfaces of described substrate contact with D type synthesis bacterium hairless protein peptide, the two sulphur rings that described synthesis bacterium hairless protein peptide contains derived from the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin are positioned at the N-end side of described ring or the 0-10 of C-end side or this two end side extra residue with containing, optionally there is the polyalkylene glycol moiety that is covalently attached to described peptide, make thus described pilin peptide covalently be attached to one or more exposed surfaces, (b) before or after described contact and combination, make described compound be covalently attached to described pilin peptide, or, be connected in the peg moiety in PEG-pilin-peptide conjugate.Preferred pilin peptide and peptide conjugate are as mentioned above.

When substrate surface has the grain boundary district of exposure, described contact procedure effectively makes described compound preferentially be positioned at the place, grain boundary district of exposure.When described material is porous or when netted, described contact procedure can effectively make described pilin peptide and hole by described material or the definite internal surface of mesh be combined.

Also disclosed is substrate, its surface bonding has (i) D type synthesis bacterium hairless protein peptide, the two sulphur rings that described synthesis bacterium hairless protein peptide contains derived from the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin are positioned at the N-end side of described ring or the 0-10 of C-end side or this two end side extra residue with containing, optionally there is the polyalkylene glycol moiety that is covalently attached to described peptide, (ii) compound, described compound is covalently attached to directly or indirectly described pilin peptide or is connected in the peg moiety in PEG-pilin peptide conjugate.

Described substrate can be metal, for example stainless steel, carbon steel, titanium, copper, brass, tin, iron, silver and magnesium and alloy thereof, and can be used as biosensor electroresponse element, wherein said compound by covalent linkage, be directly connected in described pilin peptide or by K/E spiral/spiral to being indirectly connected in the analyte receptor molecule of described pilin peptide, one in wherein said spiral is covalently attached to described pilin peptide, and another spiral is covalently attached to described compound.The stainless steel-based end, can have due to the pilin peptide with its surface bonding the electronics work content of change.

When reading following detailed description and accompanying drawing, these and other target of the present invention and feature will be more apparent.

accompanying drawing summary

Figure 1A-1D show a plurality of bacteriums belong to/kind/bacterial strain in the sequence of C-end receptors bind structural domain of IV type Pseudomonas aeruginosa (T4P) pilin;

The figure demonstration of Fig. 2 A and 2B is measured to uncoated stainless steel or with the bounding force (adhesive force) that the stainless steel that L-type K122-4 (2A) and PAO (2B) pilin peptide are coated with carries out;

The figure of Fig. 3 A and 3B shows uncoated stainless steel or the stainless electronics work content (EWF) being coated with L-type K122-4 (3A) and PAK (3B) pilin peptide;

Fig. 4 is presented at the coating that obtains in two months periods and uncoated stainless EWF measuring result;

Fig. 5 is presented at the lower coating of 800 nM load stainless power displacement (force-displacement) curve peptide and uncoated;

Fig. 6 A and 6B are presented at the result of the nano indentation test carrying out under 20 μ N (6A) and 50 μ N (6B) load;

Fig. 7 A and 7B are (being PAO in 7A, is K122-4 in 7B) and the uncoated stainless displacement measurement figure of the coating peptide under increasing load;

Fig. 8 A and 8B are uncoated (8A) and the stainless steel-based end of coating T4P17 pilin peptide (8B) and the electric conductivity AFM exterior view of the electric current between AFM tip;

Fig. 9 A-9B is measurement coating and corrosive property uncoated stainless steel surface, the stainless corrosion current (Icorr) that shows coating pilin is significantly less than uncoated stainless corrosion current (9A), be coated with pilin significantly not distinguish (9B) with uncoated stainless corrosion potential (Ecorr);

The figure of Figure 10 A and 10B shows that the stainless erosion rate that is coated with pilin, significantly lower than uncoated stainless steel (10A), compares anti-polarizability significantly higher (10B) with uncoated stainless steel;

Figure 11 shows, when pilin peptide and another peptide (in this example, leucine-zipper form E spiral or E/K coiled coil) are puted together, and the effect of corrosion inhibition reversible of the pilin peptide of being combined with metallic surface;

The photo of Figure 12 A-12C shows uncoated (12A), by the visual effect of the corrosion on (12B) of the coating of pilin peptide or the stainless steel sample that is coated with the pilin peptide with the spiral-spiral duplex that is connected in pilin;

Figure 13 shows, in the competitive binding analysis of exogenous peptide that adopts increasing amount, the pilin peptide of combination is not by from stainless steel surface replacement;

Figure 14 is XPS figure uncoated and stainless steel sample coating K-122-4 pilin peptide;

Figure 15 A shows with 15B the bounding force measuring result (15A) and the EWF power measuring result (15B) that is coated with the stainless steel sample of identical L-type and D-type pilin peptide that is coated with L-type and the stainless steel sample of D-type pilin peptide;

Figure 16 A and 16B demonstration pilin peptide (D-type) and stainless steel stent (16A) and the ability of combining closely with glass surface (16B);

The column diagram of Figure 17 A and 17B shows L-amino acid and the relevant antagonism of D-amino acid pilin to protease digestion of being combined with stainless steel sample;

The schematic diagram of Figure 18 A and 18B shows the operation of the biosensor device building according to one embodiment of the invention;

Figure 19 illustrates the analyte integrating step in biosensor, and wherein, analyte binding reagents R is directly connected in biosensor surface by pilin peptide;

Figure 20 illustrates the analyte integrating step in biosensor, and wherein, analyte binding reagents R is connected in biosensor surface by pilin peptide coiled coil mixture;

Figure 21 shows the different detection configuration in biosensor of the present invention;

Figure 22 A and 22B are before bound analyte and receptors bind and the cyclic voltammetry figure that records in biosensor device afterwards, and wherein, 22B is the enlarged view of rectangular surfaces in Figure 22 A;

Figure 23 A and 23B explanation are according to a detection of analytes device that more general embodiment builds of the present invention;

Figure 24 A-24D is column diagram, shows the combination of T4P17 pilin peptide and stainless steel (24A), crown support (24B), Foley (latex) conduit (24C) and central vein (silicone) conduit (24D);

Figure 25 A and 25B are western blotting (25A) and column diagram (25B), show that D-pilin peptide coating is on for uncoated and the coating titanium of pilin or the immunoreactive impact of the PBMC of stainless steel surface;

Figure 26 A and 26B show western blotting (26A) and column diagram (26B), show D-pilin peptide coating on for uncoated be coated with the titanium of pilin or the immunoreactive impact of the human macrophage of stainless steel surface;

Figure 27 A-27D shows that the borg sEWF of K-122-4, for EWF (27A and 27B), nano impress (27C) and erosion rate (27D) by the stainless feature of the K122-4 pilin peptide coating of various ways;

Figure 28 A-28C is the figure of the electronics work content (EWF) of the uncoated titanium of demonstration or the titanium that is coated with D type, contrary-inversion or D type-PEG pilin peptide;

Figure 29 is presented at the micro-impression effect in the lip-deep surface of titanium uncoated or coating D type, contrary-inversion or D type-PEG pilin peptide;

Figure 30 A-30E shows the micro-impression effect of bulk (bulk) on titanium plate of force level with appointment;

Figure 31 A and Figure 31 B are presented at the bounding force (Figure 31 A) of measuring on titanium plate, and the erosion rate of measuring on stainless steel plate (31B), and the two is for the plate of uncoated or coating PEG-D type pilin peptide;

Figure 32 A-32E is the titanium (32D) of uncoated titanium (31A), the titanium (32B) of coating PEG-D type pilin peptide, the titanium (32C) that is coated with the pilin peptide of contrary-inversion type, coating D type pilin peptide and is coated with contrary-inversion and the representational frictional coefficient figure of the titanium (32E) of the combination of D type pilin peptide;

Figure 33 A-33F represents the data that comparison L-pilin peptide, RI-pilin peptide and D type peptide are combined with differing materials, described material comprises stainless steel (33A, part 1-9 and 28B), titanium (33C, part A and B), urethane (33D), polysulfones (33E, part A and B) and silicone (33F);

Figure 34 shows when applying 50 mA electric currents and reach 10 minutes, in conjunction with contrasting (bound control, Ig) peptide, L-type pilin peptide (L), RI pilin peptide (RI) and the D type pilin peptide relative loss from stainless steel surface;

The figure of Figure 35 A-35D show harmless Listera ( listeria innocua) different strains (35A) and Listeria monocytogenes ( listeria monocytogenes) the bacterium of 3 bacterial strains (35B-35D) microbial film of the stainless steel plate of coating D type pilin peptide, RI pilin peptide or D+RI pilin peptide or uncoated contrast is suppressed to degree;

The figure of Figure 36 A-36F show staphylococcus not of the same race ( staphylococcus) bacterium suppresses degree to coating D type pilin peptide, RI pilin peptide or the stainless steel plate of D+RI pilin peptide or the microbial film of uncoated contrast; With

Figure 37 A-37C shows the bacteriostasis that is applied to a stainless very little PEG-D-pilin peptide.

detailed Description Of The Invention

i. definition

" pili " is the hair shape adnexa of finding on the surface of many bacteriums.

Pilin is the common name for the protein protomer of pili.

" IV type Pseudomonas aeruginosa (T4P) pilin " or " T4P pilin peptide " or " pilin peptide " refer to that Pseudomonas aeruginosa bacterium is for generation of the pili structure of motility, by making the tip of pili be attached to biological or abiotic surface and shrink pili to drag bacterium to coming to realize.All IV type Pseudomonas aeruginosas (T4P) pili all contains C-end receptor binding domain, and its oxidised form contains the two sulphur rings that can classify as 12-residue ring or 17-residue ring.Fig. 1 shows a plurality of bacterium kind Er Liuhuan district having checked order in its C-end pilin district.

" derived from two sulphur rings of the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin " refer to that its aminoacid sequence is corresponding to two sulphur rings of known bacterium two sulphur cyclic amino acids sequences, described known bacterium two sulphur cyclic amino acids sequences are for example by the definite pseudomonas aeruginosa strains PAK sequence of SEQ ID NO:4, or one of sequence that the displacement changing as the one or more amino acid in two sulphur ring sequences between two or more different bacterium kinds and/or bacterial isolates forms, one of sequence for example containing in SEQ ID NO:10, it is as four pseudomonas aeruginosa strains PAK, PAO, being combined to form of the two sulphur ring sequences of PA82935 and K-122-4.

" synthetic peptide " refers to by solid phase or the synthetic peptide forming of recombinant peptide.

" substrate being formed by stainless steel, tin, iron or titanium " means the metal base that two or more the mixture in stainless steel, tin, iron or titanium or these metals forms, or by metal or the non metallic substrate of stainless steel, tin, iron or titanium or the coating of their mixture.Substrate can contain other metal of small proportion, and the transition metal especially capable from periodictable 4-6,9-12 is listed as, comprises cobalt, nickel, copper, zinc, ruthenium, rhodium, palladium, silver, cadmium, osmium, platinum, gold, chromium (being present in stainless steel) and mercury.

" T4P pilin peptide is covalently bound with the metal base being formed by stainless steel, tin, iron or titanium " means pilin peptide and is connected in metallic surface by key, the pilin peptide that its (i) opposing is dissociated is replaced, and (ii) the vicissitudinous electronics work content of tool (EWF), shows that the surface electronic track (e-orbital) of material changes.Pilin peptide therewith the covalently bound of metalloid substrate also can be characterized by: the change of (i) change of surface adhesion, (ii) surface hardness, the change of (iii) electric conductivity and/or (iv) change (observing by the sub-spectrography of X-ray photoelectric (XPS)) at combination energy peak.

" metal base with covalently bound compound " represents to have and the covalently bound T4P pilin of substrate surface peptide, and directly or indirectly covalently bound with pilin peptide be not stainless steel, tin, iron or the titanium substrate of compound of protein another part of pilin.When compound by high-affinity in conjunction with for example, to (coiled coil leucine-slide fastener is to, vitamin H-avidin pair, etc.) while being connected with pilin peptide, wherein, a described right member and pilin peptide are covalently bound, and another member and compound are covalently bound; Compound and the indirect covalent attachment of T4P pilin peptide.

" the grain boundary district of exposure " of metal base points out existing grain boundary, that is, and and the surface region of the substrate of the interface of two polycrystalline orientations of the atoms metal of formation substrate.

" make pilin peptide preferentially be positioned the grain boundary district exposing " and refer to, pilin peptide and any compound that the is covalently attached to pilin peptide exposed surface area between Bi grain boundary district, grain boundary district has larger molecular conecentration and/or larger top coat thickness.

" L-type T4P pilin peptide " or " L-type pilin peptide " refers to the T4P pilin peptide that the amino-acid residue (L-amino-acid residue) by L-enantiomorph forms.

The T4P pilin peptide that " D type T4P pilin peptide " or " D type pilin peptide " refer to mainly or be comprised of the amino-acid residue (D-amino-acid residue) of D-enantiomorph exclusively.Specifically, D type T4P pilin peptide contains and is greater than 50% D-amino-acid residue, and is preferably D-amino acid entirely, and wherein any all the other residues are L-amino acid.D type T4P pilin peptide is protease inhibitor, and the amino acid of any L-enantiomorph in this peptide should be positioned at pilin peptide sequence, makes them can significantly not damage this protease resistant.Can determine as follows these positions: by forming and there are a series of D type pilin peptides of single L-aminoacid replacement and test monosubstituted peptide for the resistance of various proteolytic enzyme on each possible position.

" contrary-inversion T4P pilin peptide " or " contrary-inversion pilin peptide " or " RI pilin peptide " refer to the T4P pilin peptide of the reversing sequence of the amino-acid residue with D-enantiomorph, it is placed on this amino acid side chain in correct relative position, as described below.Contrary-inversion T4P pilin peptide is protease inhibitor.

" polyoxyethylene glycol " or " PEG " refers to the ethylene glycol polymer of wire or branch, and can include as few as 5 monomeric units and thousands of unit at the most, molecular weight ranges is between 0.2 kD to 500 kD or higher, and wherein specified size refers to the molecular-weight average of PEG." polyalkylene glycol moiety " refers to the polyalkylene glycol moiety (portion or moiety) of " conjugate " of pilin peptide as above and polyoxyethylene glycol.Described conjugate is covalent conjugates normally, and wherein polyalkylene glycol moiety is covalently attached to C-end and/or the-terminal amino acid of pilin peptide.

iI. pilin peptide

The obtainable various bacteriums of Figure 1A-1D display sequence information belong to/kind/sequence in the C-end pilin peptide district containing two sulphur rings of bacterial strain.The sequence providing comprises two sulphur ring sequences (start and finish with cysteine residues (C)) and is included in some cases 5 or the more residue at the most of any side of described ring.The name of single-letter amino acid is according to standard convention.In the normal oxidation form of peptide, peptide contains the disulphide bridges between cysteine residues.

The synthetic peptide adopting in the present invention comprises or derived from the one or more sequences that show in Figure 1A-1D.When sequence comprises in shown sequence, it can comprise two independent sulphur rings, or described ring can be included in any side in the N-end side of ring or C-end side or these both sides 10 at the most, preferably 5 or residue still less in addition, wherein, additional acyclic residue typically comprises or derived from one or more adjacent acyclic sequences.More generally, the sequence in ring district and acyclic district all can be by these sequences (sequence preferably in two sulphur rings with the residue of identical or almost identical quantity) are in line derived from two or more sequences.For example, in Figure 1A, corresponding to four peptides of pseudomonas aeruginosa strains PAO (SEQ ID NO:3), PAK (SEQ ID NO:4), PA82935 (SEQ ID NO:7) and K122-4 (SEQ ID NO:9), contain 14-aggressiveness (mer) two sulphur rings.From two sulphur ring sequences of four kinds of pseudomonas aeruginosa strains in Figure 1A, be in line by making, formed the sequence K/A/S/T-C-T/K/A-S/T-D/T/N-Q/V/A-D/E-E/IP/A/N-Q/M/K-F/Y-I/T/R/L-P-K/N-G/T--C-S/D/T/Q/N-K/N/D/T (SEQ ID NO:10) of combination.The peptide of this 17-residue (being also commonly referred to as T4P17) comprises 14 two sulphur ring residues, single upstream (N-end side) acyclic residue and two downstream acyclic residues.Exemplary sequence in this sequence comprises 4 the different sequences of reality that derive SEQ ID NO:10, that is, corresponding to the sequence of PAK (SEQ ID NO:3), PAO (SEQ ID NO:4), PA82935 (SEQ ID NO:7) and K-122-4 (SEQ ID NO:9).

As another example, 2 pseudomonas aeruginosa strains G7-09 (SEQ ID NO:1) and PA110594 (SEQ ID NO:2) form multiplexed sequence S/T-I-D-W-G/A-C-A/T-S-D/A-S-N-A-V/T-S/--S--G/A-T-D/A-R/Q-N/G-M/F-P/T-A/G-L/M-T/A-A-G-T/S-L/V-P-A/Q-R/E-F-A-P-S/A-E/Q-C-R (SEQ ID NO:21).

Once select pilin peptide sequence, it can synthesize by re-constituted established law or the solid-phase synthesis of standard.For example, by the recombinant expressed E-spiral of pRLD-E plasmid (E-coil) PAK (128-144) ox, wherein, utilize the in-frame montage PAK of synthetic oligonucleotide (128-144) ox DNA sequence dna and E-spiral and according to known technology intestinal bacteria ( e. Coli) express (referring to such as people such as Giltner, Mol. Microbiology (2006) 59 (4): 1083 and the reference wherein quoted) in bacterial strain BL-21.By the expressed peptide of metal affinity chromatography purifying, by mass spectroscopy and the order-checking of N-terminal protein, confirm the formation of purity and disulphide bridges.

In a general embodiment of the present invention, pilin peptide comprises D-amino acid, and as mentioned above, can contain one or more L-amino acid, as long as L-amino acid is minority part (being less than 50% residue) and protease resistant that can obvious damage peptide.At pilin peptide, comprise that the amino acid whose object of D-is to increase the proteoclastic resistance that peptide causes for one or more proteolytic enzyme that may be touched by peptide.For example, Rhodopseudomonas bacterium has a series of proteolytic enzyme, comprises elastoser, metalloprotease and typical trypsin-like serine protease, needs its target Methionin and/or arginine residues in peptide cracking.Therefore, can synthesis bacterium hairless protein peptide to contain D-Lys, for example K136 in K122-4 pilin peptide and K140 place.Preferably, in preparation, proteolytic enzyme as much as possible is had in the peptide of resistance, pilin peptide should be formed by D-amino acid completely.Can pass through conventional solid phase method, in synthetic, utilize the D-that activates or L-type amino-acid reagent to form all by D-amino acid, to be formed or by the pilin peptide of D-and the amino acid whose compositions of mixtures of L-.(referring to for example Guichard, G., waits people, Proc. Nat. Acad. Sci. USA. the 91st volume, 9765-9769 page, in October, 1994).

As what it will be appreciated that below, to compare with L-type pilin peptide, D type pilin peptide has considerable advantage when as surface coated agent.First, although L-type T4P pilin peptide effectively suppresses the biofilm formation that pseudomonas causes, but the biofilm formation that D type pilin peptide causes for pseudomonas provides better inhibition, and for example, provide significantly stronger inhibition for the biofilm formation due to non-pseudomonas (Listera and staphylococcus).Secondly, although L-type T4P pilin peptide with high-affinity effectively in conjunction with various materials, comprise various metals, polymkeric substance, silicate and pottery, but when material is exposed to proteolytic enzyme, with the electro-conductive material in the situation that when electric current is applied to material, D type peptide provides significantly more stable combination conventionally.

In another general embodiment, pilin peptide comprise with opposite sequence direction, be carboxyl terminal to the synthetic D-amino acid of amine extreme direction, with produce so-called contrary-inversion (RI) pilin peptide.Contrary-inversion (RI) type pilin peptide also has advantages of stronger protease resistant, therefore and be favourable in described application in this article, the material that is coated with pilin in described application is exposed to proteolytic enzyme, for example, in coenocorrelation or in standing the environment of bacterial growth.The method of synthetic RI type peptide has detailed description: Fletcher in for example with Publication about Document, M.D. and Campbell, M.M., Partially Modified Retro-inverso Peptides:Development, Synthesis, and Conformational Behavior, Chem Rev, 1998,98:763-795, it is incorporated herein by reference.

As what it will be appreciated that below, to compare with L-type pilin peptide, contrary-inversion pilin peptide also has considerable advantage as surface coated agent.First, the biofilm formation that they cause for pseudomonas provides better inhibition, and for example, provide significantly stronger inhibition for the biofilm formation due to non-pseudomonas (Listera and staphylococcus), although generally speaking, compare with RI peptide, D type pilin peptide produces stronger restraining effect to biofilm formation.Secondly, although L-type T4P pilin peptide with high-affinity effectively in conjunction with stainless steel (by being characterized by the electronics work content (EWF) of increase, larger hardness and covalency-sample key of corrosion resistance), but have been found that contrary-inversion pilin peptide produces significantly higher steel EWF (producing higher than L-type peptide), and significantly increased material hardness, also show corrosion resistance larger (with respect to viewed with L-type peptide).

In a further embodiment, by the pilin peptide of pilin peptide and preferred D type or contrary-inversion type, be conjugated to polyethylene glycol polymer wire or branched chain, conventionally by the end of covalently bound polyoxyethylene glycol wire or branched polymer and the N-terminal amine of peptide and/or C-end acid groups, use conventional peptide coupling method, for example, for form amido linkage, ester bond, ehter bond or disulfide linkage or the key that contains activating group between peptide and peg moiety.It is commercially available available being used for the PEG puting together with protein or the PEG with suitable reactions group, for example, from PEGTech, Analytical Ventura and Thermo Scientific (PEG of branch) and activating group can be maleimides (being generally used for and following radical reaction: peptide sulfydryl, vinyl sulphone, propionic aldehyde, pyridine two sulfones, NHS ester or iodo-acid amide) for example; Can, by known linked reaction, the PEG with terminal amine, acid, hydroxyl or sulfhydryl reactive group be coupled to amine, acid, hydroxyl or the methylthio group of peptide.PEG at one end has inertia group conventionally, and for example methoxyl group, has activating group or reactive group at the other end.The selection magnitude range of the typical mean molecular weight of polymeric reagent is between 5kDa to 40kDa, and be equivalent to has 100-1 in polymkeric substance, and 000 repeating unit, although smaller or greater PEG is also suitable.According to standard P EG coupling method, by activated PEG reagent, protein carry out PEGization.

The advantage of PEG-D-pilin peptide or PEG-RI-pilin peptide below will seen.Generally speaking, the D type of PEGization form or RI pilin peptide provide above D-or the mentioned advantage of RI type peptide for non-PEGization form, but additional advantage is for significantly reducing frictional coefficient and larger the biofilm formation being suppressed on coating surface.

iII. process metal surfaces

On the one hand, the present invention includes the improving one's methods of stainless steel, tin, iron or titanium metal material that treat surface has the grain boundary district of exposure, to reduce the erosion rate of material.The method can be individually or with multiple other anticorrosive method in a kind of (for example passivation) be used in combination.Metallic substance can have single exposed surface (it has grain boundary district) or a plurality of pending outside surface, or contain from outer surface can and hole or inner mesh.Be appreciated that the method is applicable to standing any processing of any stainless steel, tin, iron or the titanium metal material of chemical corrosion, described chemical corrosion is for example in the atmosphere of oxidation or by for example, contacting with corrosive liquid (alkalescence or acidic liquid).

In implementing the method, can first wash metallic substance one or many, for example, in ethanol bath, to remove pollutent.Then, effectively make under the covalently bound condition of exposed surface of pilin and material, material to be contacted with pilin solution.In a typical treatment process, material is positioned over to peptide or conjugate concentration is for example, for example, for example, in the pilin peptide or the solution of PEG-pilin-peptide conjugate in approaching the aqueous buffer solution (phosphate-buffered saline) of neutral pH (pH 7) of 2 μ g/mL to 50 μ g/mL pilins (10 μ g/mL), and makes material contact for some time (for example 5-120 minute) with this solution until suitable pilin peptide coating forms.

Or, can use pilin solution spray material to be coated, and make the duration of contact (for example 5-120 minute) of itself and spray solution contact need in high humidity environment.

In a further embodiment, pilin coating is put on to the selected areas of metallic surface, for example, in micro-manufacture (microfabrication) operation, or optionally peptide is put on to the grain boundary district exposing on material.In this embodiment, in regiospecificity mode (for example, by ink-jet printer or similar means), peptide solution is delivered to the exposed surface of material.

iIIA. the change for the treatment of process and metallic surface character

Process metal surfaces has been described to strengthen the illustrative methods of its corrosion resistance and the research of carrying out supporting the present invention in this part, described studies have shown that, except the corrosion resistance strengthening, (i) bounding force of treated surface reduces, (ii) the electronics work content of treated surface changes, (iii) hardness of treated surface increases, and (iv) electric conductivity reduces, and (v) at least bimestrial period undercoat stable.Except as otherwise noted, otherwise carry out with L-type pilin peptide in this part and the research reported in part IIIB and IIIC.The research of reporting in part IIID and result are to carry out with the PEG-conjugate of D type and contrary-inversion pilin peptide and D-and RI pilin peptide.

sample preparationcommercial grade 304 2B finish plates (finish plate) (20 specification) stainless steel plates (1 mm is thick) are cut into the sample that is of a size of 1 cm x 1 cm.Sample 1140 ℃, in air, anneal 1 hour and cooling in air.The sand paper glazed surface of use 120,240,320,400,600 and 800# granularity, carries out final polishing with 1200# grit paper subsequently.

Use above-mentioned polishing scheme polishing to be of a size of aluminium and the stainless steel sample of 1 cm x 1 cm x 1 cm.These samples were not all annealed before polishing.

by peptide or monomer pilin coated sampleuse the commercially available dish soap of washing that stainless steel and aluminium sample are washed 1 minute, use subsequently distilled water flushing.Then follow mild stirring that sample is immersed in 95% ethanol 15 minutes, with distilled water flushing, then immerse in reagent grade acetone 1 minute.With distilled water flushing sample 5 times, make subsequently it air-dry.Sample immersed in phosphate-buffered saline (PBS) solution contain 10 μ g/mL peptides or peptide conjugate or monomer pilin and follow mild stirring in room temperature (RT) incubation 1 hour.Remove solution, with distilled water wash sample 6 times and make sample air-dry.

Use the clean carbon steel sample of such scheme, but instead with 100% washed with methanol sample, exist side by side and be about to sample immerse the air corrosion fast to prevent from causing in 100% methyl alcohol when being exposed to water washing with acetone step after.Peptide or peptide conjugate are dissolved in 100% methyl alcohol, and the ultimate density of 10 μ g/mL is for submergence carbon steel sample.Follow mild stirring in RT by sample incubation 1 hour.With 100% methanol wash sample 6 times and make it air-dry.

bounding force is measureduse atomic force microscope (AFM) to measure the bounding force between the surface that tip radius is the most advanced and sophisticated and coating peptide of the AFM silicon nitride of coating Au of standard of 50-70 nm.In order to determine the bounding force between the surface that AFM is most advanced and sophisticated and be coated with, with " contact " pattern using AFM.Make the most advanced and sophisticated surface that approaches, make their contacts, the deflection (deflection) of measuring cantilever (cantilever) when tip is pulled away to surface.By the total amount of laser beam detection deflection, it reflects bounding force.If the spring constant of cantilever is known, can be according to beam deflection (beam deflection) and quantitative assay bounding force.In this research, cantilever spring constant is 0.06 N/m.To each experiment, each sample obtains 20 to 50 bounding force measuring results.

The result of the bounding force research of the stainless steel sample of coating K122-4 or PAO pilin peptide is plotted in respectively in Fig. 2 A and 2B.As shown in Figure 2 A, the bounding force of the metal of coating concentrates in the scope of about 5-40 nN (Na Niudun), mean value approximately 20 nN, and compared to uncoated sample, its bounding force concentrates on about 40-75 nN, mean value approximately 60 nN.By PAO pilin coating, obtain similar result.For example, because bounding force is the reflection of electronically active (Van der Waals (Van de Walls) interacts), deducibility goes out peptide coating and plays the effect of sheltering metallic surface electronic shell.

The similar bounding force of the aluminium sheet of coating peptide is measured and shown, the bounding force between coating and uncoated plate is almost as broad as long.

work content is measuredwith SKP370, scan Kelvin probe (Scanning Kelvin Probe) and measure as usual electronics work content (EWF) coating and uncoated stainless steel sample.Use capacitance-type vibration probe and operate this technology by frequency sweep backing potential (swept backing potential), measuring scan-probe poor with reference to the work content between tip and sample surfaces.Studied sample be as in bonding research, use those, difference has been also to check the sample with the coating of PAK pilin peptide.

Result of study is plotted in Fig. 3 A in (about uncoated and sample coating K122-4 pilin peptide) and Fig. 3 B (about uncoated and the sample that is coated with PAO-and PAK pilin peptide).For all three kinds of coatings, pilin peptide coating has improved at least about 0.5 eV surperficial EWF, reaches the end value of about 5eV.

The similar EWF of the aluminium sheet of coating peptide is measured and shown, the EWF between coating and uncoated plate is almost as broad as long.

the stability of peptide coatingfig. 4 shows being coated with the measuring result of the sample of the coating K122-4 pilin peptide obtaining in the time limit of latter 2 months.The sample of observing coating in this study period of 2 months has higher EFW with respect to uncoated sheet, shows at least bimestrial coating stability.

nano impress/hardnessuse triboscope (Hysitron, Minneapolis, USA) to check the change of the mechanical property of the sample that is coated with peptide.Triboscope is the combination of nano-machine probe and AFM.This probe is diamond cone bodily form Vickers hardness tester pressure head, has the displacement resolution of the nominal radius of 150nm, the force sensitivity of 100 nN (force sensitivity) and 0.2nm.In nano impress process, for each impression, obtain power-depth curve, from this curve, obtain the most advanced and sophisticated total depth displacement that enters sample surfaces.Use the power of 50 to 800 μ N to carry out nano indentation test.To five power-depth curves of each power Load obtaining.

In Fig. 5, show (dark color) and the uncoated force-displacement curve of (light color) stainless steel under the load range of 50 to 800 μ N of coating peptide.Total displacement under 800 μ Ν loads is presented at the top of figure, and for the sheet of coating, this is worth within the scope of 45-55nm, and for uncoated sheet, this is worth at about 90-95nm.Based on this test, the hardness of the sheet of coating is almost the twice of uncoated sheet.More generally, coating makes the hardness of stainless steel, tin, iron or surface of metal titanium increase at least about 20% effectively, preferably at least about 30%, and at the most 50% or higher.

Fig. 6 A and 6B draw displacement coating and uncoated sheet nano impress of generation under 20 μ N (6A) and 50 μ N (6B).With the data consistent from Fig. 5, the high about 20%-100% of sheet that the hardness ratio of the sheet of coating is uncoated.

The research of same type is plotted in Fig. 7 A and 7B, figure (7A) for coating PAO's and within the scope of the power of 50-800 μ N, figure (7B) for coating K122-4's and within the scope of the power of 50-400 μ N, result is substantially the same.In both of these case, coating pilin peptide almost makes the surface hardness of metal sample double.

To the similar nano indentation test demonstration of the aluminium sheet of coating peptide, the surface hardness between coating and uncoated plate does not almost change.

the electric conductivity increasingelectric conductivity is the measuring of ability of material conducts electricity.A standard method of surface measurements electric conductivity is used atomic force microscope (AFM) to measure the electric current that flow to AFM tip under the low voltage potential bias voltage (potential bias) of regulation from lip-deep specific position.AFM is quantitatively shown as particular color by the electric current between surface and tip (in PA), and for stainless steel plate uncoated and coating pilin, represents respectively with gray shades different in Fig. 8 A and 8B.Conventionally, be deep to the large extremely less electric current (28.0 to 24.5pA) of more shallow shadow representation.From this two width figure, observe, the surface region of the uncoated stainless steel sample in Fig. 8 A alters a great deal and has the leading dark-shaded that represents high current on sample surfaces, and the surface region that is coated with the sample of pilin in Fig. 8 B be mainly low electrical conductivity and electric conductivity figure significantly more even.These results with from the EWF data consistent of Fig. 3 A and 3B, show that the material that is coated with pilin has significantly higher metal work content (extracting measuring of the required merit of surface electronic).

corrosion resistancethere are multiple technologies to can be used for corrosion resistance or the susceptibility-to-corrosion on research material surface.A kind of method is to measure the electric current that passes metal sheet under fixing current potential.Between redox form back and forth, larger electric current shows larger corrosion potentiality to the electric current reflection surface electronic recording.Figure in Fig. 9 A shows the electric current (Icorr) that 304 2B finish plates (20 specification) stainless steel plate of as above preparation (uncoated or coating K-122-4 pilin peptide) is recorded.Result shows that the Icorr of the plate being coated with is significantly lower, shows that corrosion resistance is higher.

May be asked about, the current potential (Ecorr) when first whether the Icorr difference of observing in above research start to flow through metallic surface with electric current is relevant.Current potential (Ecorr) while first starting to flow by the electric current in observation metal is studied this problem.Result of study (being presented in Fig. 9 B) shows, coating have similar Ecorr value with uncoated metal sample, show the Icorr value difference of seeing in Fig. 9 A different be not that difference by voltage potential response between two samples causes.

In Figure 10 A, drawn coating erosion rate measuring result K-122-4 and uncoated sample (to measure in mil (mil)/year (mpy)).Result is consistent with the difference of the Icorr seeing in Fig. 9 A.Particularly, when comparing mean rate, pilin peptide coating shows has reduced over three times erosion rate.

In corrosion monitoring, another widely used technology is anti-polarizability, is defined as the current potential current density slope of a curve under freely corroding potential, and obtaining can be by the known mathematical relation resistance value Rp relevant to corrosion current.Figure 10 B draws coating and uncoated stainless Rp value, and show peptide coating significantly improves the Rp measuring as corrosion resistance.

Interestingly, when pilin is puted together with the another kind of peptide with strong dipole and/or high charge density, in this example, described another kind of peptide is leucine zipper type E spiral or identical E spiral (it is in conjunction with the K spiral of the opposite charges of E spiral/K spiral centering), the effect reversible of pilin peptide in suppressing corrosion.As shown in figure 11, the erosion rate of uncoated stainless steel sample is significantly lower than the sample with the pilin-E of combination or the pilin of E/K spiral form.

To the visual effect of the corrosion test of various stainless steel samples discussed above referring to Figure 12 A-12C.In this research, sample or uncoated (12B) or the pilin peptide of puting together is coated with pilin peptide (12A) or with E/K coiled coil.In each case, sample experiences above-mentioned corrosion test in rare salts solution.Compare with uncoated plate, the plate of coating pilin shows minimum surface corrosion, and pilin-conjugate coating seems significantly to add deep-etching.

In a word, with synthesis bacterium hairless protein peptide coating metal for example stainless steel, tin, iron or titanium, effectively increase hardness and the corrosion resistance of metallic surface, described synthesis bacterium hairless protein peptide contains derived from two sulphur rings of the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin and contains and is positioned at the N-end side of described ring or the 0-10 of C-end side or this two end side, 0-5 extra residue preferably.The corrosion resistance increasing is by changing, and the change that for example the electronics work content of metallic surface is increased to Shao0.2 EFW unit and above-mentioned Icorr, erosion rate and Rp value proves.The hardness improving has reduced at least 20% by nano impress to be proved, described nano impress impacts metallic surface by the tip by atomic force microscope with given force and produces.

Other metal of considering in described method is that periodic table of elements 4-6 is capable, the transition metal of 9-12 row, comprise cobalt, nickel, copper, zinc, ruthenium, rhodium, palladium, silver, cadmium, osmium, platinum, gold and mercury, their mixture and alloy, metalloid silicon and germanium and their oxide compound.

the frictional coefficient reducing

According to currently known methods, for example in 2007 J. Phys.:Conf Ser 61 51, report, use atomic force microscope (AFM) to measure frictional coefficient.In brief, for example, with given force (500 mN) socle girder of AFM (cantilever beam) is applied to surface, and crosses over test surfaces and move.The amount of deflection of beam just provides the tolerance of the frictional force that beam experiences, and lower frictional coefficient produces lower deflection.The deflection overview of beam whole mobile alignment from the teeth outwards just provides the tolerance of frictional coefficient, little and more uniform deflection represents low coefficient, and higher and more irregular overview represents higher beam deflection.

iIIB. the covalently bound extra evidence of pilin peptide and treated metal

The electronics work content of the metal of coating changes and corrosion resistance enhancing shows, pilin peptide has changed the surperficial electronic property of coating, the formation of covalent linkage between hint peptide and metal, and it has changed the unbound electron track of metal.This discovery additionallysupport is from the peptide substitutability analysis of reporting in this branch and the sub-spectrography of X-ray photoelectric (XPS) research.

the substitutability analysis of peptide/pilinan indication of the covalent interaction between compound and substrate is can not replace compound from substrate during incubation mixture under the compound of solute form exists.At this, the ability of the pilin peptide that the displacement of Study of Exogenous sex fimbria protein peptide is combined with stainless steel surface.Clean as described above thick commercial grade 304 2B finish plates (20 specification) stainless steel plate of 1 mm.These plates are not annealed or polishing.These plates are assembled in 96 hole Schleicher and Schuell Minifold TM System (Mandel Scientific).The pili that 50 microlitres are contained to the biotinylated PAK peptide of 10 μ g/mL or biotinylated purifying albumensolution add in hand-hole (5 replicate(determination)s) and under RT, follow mild stirring by this collecting tubule (manifold) incubation 1 hour.With 1x PBS washing hole six times.To adding the unlabelled PAK peptide of increasing amount (0 to 10 μ g/mL) in replicate(determination) hole and following mild stirring by this steel collecting tubule incubation 1 hour under RT.Use subsequently PBS washing hole six times.Use the biotinylated peptide of streptavidin-horseradish peroxidase (HRP) assessment combination or the displacement of pilin.By streptavidin-HRP (Sigma) 1/500 dilution, every hole add 100 μ L and under RT by this collecting tubule incubation 1 hour.Every hole adds 150 microlitre development buffer (to contain 1 mM 2, the 0.01 M sodium citrate buffer solution of 2'-azino-bis--(3-Ethylbenzyl thiazoline-6-sulfonic acid) di-ammonium salts (ABTS) (Sigma) He 0.03% (v/v) hydrogen peroxide, pH 4.2).Under RT, follow mild stirring by this collecting tubule incubation 10 minutes.Reaction soln is transferred in 96 hole flat-bottom microtiter plates (Corning), uses FLUOstar OPTIMA to read the absorbancy that plate instrument (BMG LABTECH) is determined at 405 nm places.

As shown in the data of drawing in Figure 13, even when the solubility pilin peptide of higher concentration, there is no the loss from measurable combination pilin peptide of metallic surface, prove that the pilin peptide of combination and unconjugated peptide balance each other, show that the covalent linkage between peptide and metallic surface connects yet.Covalently bound other evidence of peptide and metallic surface is provided by following corrosion resistance research.

xPS featurethe sub-spectrography of X-ray photoelectric (XPS) is the quantitative spectrography technology of measuring the electronic state of the element existing in elementary composition and material.Kinetic energy and the quantity of by irradiate material with X-bundle of rays, measuring the electronics of escaping from top 1 to 10 nm of analyzed material obtain XPS spectrum simultaneously.XPS needs ultrahigh vacuum(HHV) (UHV) condition.

Use Axis-165 spectrograph (Kratos Analytical) check from photoemissive electronics uncoated and sample coating pilin.From the spectrum of these two kinds of sample ejected electron, see Figure 14.As directed, the sample of coating pilin contains in conjunction with being about 100 and two unique peaks of 150eV, and they do not exist in uncoated sample.A possibility is that these two peaks represent due to the electronics combination of puting together the significantly sulphur-metallic bond of red shift.The evidence that there is no N or O and melts combine, show covalency possible between pilin and metal (shared electron) key interact be with pilin peptide in one or two in two sulphur atoms.

iIIC. grain boundary effect

Grain boundary is the interface between two crystal grain or crystallite in polycrystalline material.Grain boundary is the defect in crystalline structure, and is easy to reduce electricity and the thermal conductivity of material.High interfacial energy in most of grain boundaries and weak combination often make them become the preferential site of corroding the phase that starts and make new advances from solid precipitation.

Because grain boundary can be used as the starting point of corrosion, so, determine that pilin peptide is combined with metallic surface whether preferentially to occur in site, grain boundary be interesting.In order to study this problem, the above-mentioned bounding force that use is coated with to the AFM tip of pilin-peptide is studied the further meticulous bounding force effect of locating with grain boundary in research crystal grain that turns to.The stainless steel plate of the peptide coating of PAO pilin peptide or out of order with having (scrambled) PAO aminoacid sequence for " test " in this research and " contrast " surface.For each sample, measure the bounding force of locating with grain boundary in crystal grain.

As shown in the result providing in following table 1, in grain boundary, the bounding force of pilin peptide is than little approximately 20 nN of bounding force with the same material of out of order sequence, and at place, grain boundary, little approximately 43 nN of bounding force.This result shows, or pilin peptide is preferentially positioned place, grain boundary, that is, peptide has larger coat-thickness at place, grain boundary, or the pilin of par is combined in place, grain boundary and produces larger bounding force effect.In either case, these data are soluble by making pilin peptide be combined the size of viewed anticorrosive effect with metallic surface.

Table 1: the effect of grain boundary district to bounding force

AF M is most advanced and sophisticated	Rival	Bounding force in crystal grain (nN)	The bounding force (nN) at place, grain boundary	The increase multiple at place, grain boundary
					Coiled coil-PAK (128-144) ox	PAO(128-144)ox	39.5 ± 9.4	44.5 ± 11.6	1.13
Coiled coil-PAK (128-144) ox	PAO (128-144) ox_ is out of order	59.7 ± 8.4	87.5 ± 14.2	1.47
					Be attributable to PAK (128-144) ox	?	20.2 ± 17.9	43.0 ± 25.8	2.12

iIID. by D-and RI-type pilin, carry out the combination of pilin peptide

In order to check D-and RI-type pilin peptide and multiple material, comprise that various metals comprises the ability of (stainless steel) and polymkeric substance combination, and the properties of materials of coating pilin, synthetic D-tests with the identical pilin peptide of RI-type K122-4 pilin peptide relative L-type.

In a research, with L-type pilin peptide (three different batches), D-type pilin or RI-type pilin coating stainless steel plate, a plate is not coated with.First check the change of the bounding force of described plate, be similar to the above research of reporting for Fig. 2 A and 2B.As shown in Figure 15 A, to triformed pilin peptide, all see that bounding force significantly reduces with respect to uncoated plate.

The EWF measuring result of six samples to identical (as above for carrying out as described in Fig. 3 A-3B) is plotted in Figure 15 B.Interestingly, D-amino acid form provides than any one (comprising uncoated plate) in other five plates remarkable low EWF, and RI pilin peptide has significantly increased the EWF of steel, higher than the viewed EWF of stainless steel with the L-type pilin peptide of combination.These results also see in Figure 27 A, the figure illustrates for (being called borg-K122SS) with L-type K122-4 pilin peptide coating or by (being called borg-K122SS-D-amino) of the coating of D type K122-4 pilin peptide or (be called borg-K122SS contrary-inversion) measured EWF value of stainless steel (Borg, Grade 304 stainless steels) of being coated with contrary-inversion K122-4 pilin peptide.As desired, contrary-resistance to tryptic digestion of inversion pilin peptide (all seeing Figure 27 B) and significantly increase the hardness (Figure 27 C) of steel.Because EWF is the energy level of surface electronic and the tolerance of the ability that these electronics react with other material, so the EWF raising has reflected the increase of corrosion resistance conventionally.Shown in Figure 27 D, studies confirm that this point, prove borg-K122SS contrary-the anticorrosive speed of inversion is uncoated stainless approximately 50%.

Result shows that D-and RI-type pilin peptide can interact to change mode and the stainless steel plate of the electronic property of metallic surface, wherein contrary-inversion type provides than the remarkable high EWF of L-type pilin peptide, and provides than the high a lot of EWF of D type pilin peptide.

By peptide relatively, with uncoated or with the combination of stainless steel stent biotinylated control peptide (out of order pilin sequence or the non-binding district of pilin sequence) coating or that be coated with biotinylated D-type pilin peptide, study the ability that D-type pilin and stainless steel are combined closely.By first in 37 ^oc, with 3% SDS solution washing surface, carries out other several washing subsequently in PBS, measures the amount of the peptide of being combined with each rack surface.Surface incubation together with streptavidin-HRP (horseradish peroxidase) by washing, is then exposed to ABTS substrate, reads the absorbancy at 405nm place.The D-type pilin peptide of being combined with stainless steel stent as shown in Figure 16 A, is about the twice of control peptide.

In order to study and the D-type pilin peptide of the melts combine zymoprotein hydrolysis resistance with respect to the L-type pilin peptide with melts combine, with L-type peptide, D-type peptide and contrast (out of order pilin sequence), be coated with in duplicate stainless steel plate.Then make trypsinase from sample of every two parts and 0.25% concentration, EDTA 1 mM, pH 7.4 37 ^oc is incubation 60 minutes together.Afterwards, by the combination albumen of above-mentioned HRT analytical method analytic sample.

Figure 17 A is presented at the binding peptide level that is exposed to trypsinase L-type pilin before and afterwards.Just as can be seen, by protease treatment, remove the pilin peptide over half.By contrast, in conjunction with the amount of D-type pilin peptide be not substantially subject to the impact (Figure 17 B) of protease digestion.This result proves that (i) L-type peptide and stainless covalent attachment do not give the protective effect of protease inhibitor digestion, and (ii) the D-type pilin peptide of combination is substantially protected and avoids zymoprotein hydrolysis.

For the significant advantage that additionally studies have shown that following aspect of supporting that the present invention carries out: D type and contrary-inversion pilin peptide provide than the higher binding affinity of L-type pilin peptide and proteolysis resistance.These advantages are outstanding especially for D type pilin peptide, as being about to see.

Figure 28 A-28C shows that different D and RI type pilin peptide and peptide-PEG conjugate are to unpolished (Figure 28 A) with the effect (Figure 28 B and 28C) on the titanium surface of 600 granularity polishings.Just as can be seen, the K122 pilin peptide of D-and RI type all can increase metallic surface EWF with measuring, and wherein D type all obtains maximum EWF for these two kinds of titanium surfaces increases.PEG-D-pilin peptide (Figure 28 C) has increased EWF, but degree is lower than the D type peptide of non-PEGization.

Nanoindentation to the titanium surface of the D-with different and Ri type pilin peptide (comprising PEG-D-pilin peptide) coating is shown in Figure 29.Compare with uncoated titanium, RI and D type peptide (comprising the D type peptide that PEG puts together) all demonstrate significant surface hardening.PAO-K1301 peptide is the L-type pilin peptide that (according to the numbering plan of PilA albumen) has amino acid mutation in residue position 130, and wherein natural lysine residue is replaced by Isoleucine residue.

In order to prove that the surface hardness effect shown in Figure 29 is limited to surface, not for example bulk metal effect (bulk-metal effect), to using the steel surface of the pilin peptide identical with Figure 29 coating to carry out micro-impression measurement, but as shown in Figure 30 A-30E with high impact forces more.During just as can be seen, with higher force, hardness has no variation.

Figure 31 A shows that PEG-D-pilin peptide coating is to the effect of the bounding force on titanium plate (Figure 31 A) with to the effect of the erosion rate of steel plate (Figure 31 B).The D type peptide effects on surface of PEGization is bonding almost less than effect, but can significantly reduce the erosion rate of steel plate.

In order to prove that the pilin peptide of PEGization significantly reduces the metal of coating (in this example, titanium plate) frictional coefficient, to uncoated titanium surface with the titanium that different pilin peptides are coated with, carry out AFM friction coefficient measurement, as mentioned above, the results are shown in Figure 32A-32E.Each figure is the representative diagram of choosing in 6 different figure of every kind of coating generation.Uncoated titanium surface provides high and irregular displacement, as shown in the figure of Figure 32 A.In the titanium plate with the coating of PEG-D-peptide, observe minimum frictional coefficient, see the figure of Figure 32 B.This figure is characterised in that little in studied surface line (surface line) and displacement quite uniformly.Although the peptide of the RI type of non--PEGization (Figure 32 C) and D type (32D) have significantly reduced frictional coefficient, this two width figure does not demonstrate the minimizing of the frictional coefficient suitable with PEG-D-peptide.When application D and RI type non--during the peptide of PEGization, observe some improvement (Figure 32 E), show that D-and RI type are attached to the different sites on metallic surface.Yet, combination non--peptide of PEGization still provides and more irregular displacement higher than independent PEG-D-peptide.

Frame 2-9 in Figure 33 A is the fluorescence microscopy figure (showing with 20X in frame 1) of stainless steel stent, and its surface has been coated with control peptide (frame 2 and 3), L-peptide (frame 4 and 5), RI pilin peptide (frame 6 and 7) and D type pilin peptide (frame 8 and 9).Just as can be seen, D-peptide has retained hyperfluorescenceZeng Yongminggaoyingguang after incubation together with whole blood, expresses maximum proteolysis resistance.Figure 33 B shows the point of the peptide/albumen detecting with streptavidin-horseradish peroxidase (being connected with biotinylated peptide/albumen).On stainless steel, only there are D type and Ri pilin peptide opposing Proteinase K to process.Compare with L-type pilin peptide, these two kinds of pilin peptides also can more effectively be attached to the stainless steel-based end.When L-type, D type and RI pilin peptide are attached to titanium, observe similar results (33C), wherein right figure A shows that D type peptide has maximum Proteinase K resistance when being attached to titanium substrate.Combination at the bottom of D type and RI pilin peptide and polyurethane-base, can be observed similar results, and as shown in Figure 33 D, it demonstrates with control peptide and compares, the combination level of D type and RI pilin peptide and substrate.Although do not show herein, L-type peptide demonstrates weak combination relative to urethane.

When these 3 kinds of pilin peptide forms are attached to polysulfones dialysis membrane and be exposed to subsequently different destruction processing, obtain similar results.See Figure 33 E frame A, observe D type and RI pilin peptide is significantly larger than the tryptic digestion resistance of L-type peptide.Figure 33 E frame B is presented at the relative quantity of processing and boiling 10 minutes binding peptides afterwards with SDS, Proteinase K.In all cases, D type peptide is the most stable on being attached to film time, and L-type is the most unsettled, the stability in the middle of RI peptide demonstrates.For these the 3 kinds of peptides at the bottom of being attached to silicone base, also can be observed this pattern (Figure 33 F).

Interesting is the relative stability of these 3 kinds of pilin peptide forms of research, when the stainless steel-based end of their institute's combinations experiences gentle electric current.In a research, it the results are shown in Figure 34, applies 18V, and 50 mA electric currents reach 10 minutes by the stainless steel-based end, causes control peptide, L-type peptide and RI peptide actual consumption, but D type peptide demonstrates binding peptide loss seldom or not loss.When reduced-current (30 and 40 mA, at same time in the time limit), obtain similar results (data do not show).These results are appropriate especially to multiple analytical form as described below, and wherein the acceptor molecule of binding partner is attached to metal base by pilin peptide.When making substrate be exposed to gentle electric current, D type pilin peptide retains the ability of its combination, allow substrate to dispose the material of non-specific binding, by applying medium current by substrate, the remarkable loss analysis thing signal relevant to receptors bind not, described acceptor is anchored in substrate by D type pilin peptide.

iIIE. related application

According to another aspect of the present invention, also can utilize pilin peptide in conjunction with and increase the ability of the hardness of coating material, with surface hardening other materials for example sheet glass or automotive safety glass.In this application, clean glass surface is contacted under certain condition with pilin peptide, described condition makes surface coated have pilin layer effectively.Use HRP to analyze the amount of as above measuring the protein of being combined with substrate.As shown in Figure 16 B, D-type peptide and glass surface are combined closely, though the effect of removing that opposing is processed by SDS, and the pilin peptide of being combined with stainless steel stent is the approximately twice of control peptide.

In Another application, surface treatment is for strengthening the machine oilness of the metallic surface of the coating of moving contact mutually.At this, target machine assembly is by as above making component exposed come pre-treatment to strengthen surface lubrication in pilin peptide under the condition forming covalently bound pilin coating.Or, the solution of pilin peptide can be put on to the surface in contact of machine during operation or during temporary transient shutdown, to maintain the larger oilness of machine component in machine operation process.

iV. the metal base and the biosensor device that are coated with

This part considers that the present invention is applied to diagnostic device, wherein, makes analyte-specificity target compound (for example acceptor) be attached to and be detected surface by pilin peptide of the present invention or peptide conjugate.When detecting surface and be the metal with pilin peptide covalent attachment (by the interaction of electrons with metallic surface), described device can electronic biosensor pattern work, as described below.

the metal base IVA. with covalently bound compound

The party's face of the present invention comprises metal base, compound (for example acceptor) is covalently bound at substrate surface and described metal base in the following manner, (i) as detailed above, pilin peptide and substrate covalently bound, and compound and pilin is directly or indirectly covalently bound, that is, by pilin-compound conjugate, or be directly connected with peg moiety.The following substrate that forms coating: by first make unconjugated pilin peptide or conjugate and metallic surface is adhered to, make subsequently the pilin of compound and combination or PEG covalently bound, or by first forming pilin-compound conjugate or PEG-pilin-compound conjugate, making this conjugate be combined (as above for described in unconjugated pilin peptide) with metallic surface subsequently.Making the covalently bound method of compound and pilin peptide, for example, by through amine or the direct chemical coupling of carboxyl or use difunctional coupling reagent, is well-known.When compound itself is peptide, can as fusion rotein, form pilin-compound conjugate by re-constituted established law or solid-phase synthesis.Combination due to pilin and metallic surface, the substrate of coating has changed surface electronic character, and the research (describing in detail below) of supporting the present invention and carrying out shows, by analyte associated molecule with the combination of the compound of surface bonding, regulate the electric current by substrate surface, make to change to record this type of binding events by the electric current through substrate.As what it will be appreciated that below, can also make compound indirectly be covalently attached to substrate, for example, by E/K coiled coil mixture.As what can the data from Figure 33 A-33F understand, D type pilin peptide is preferred in this application, considers its more stable binding characteristic to various base materials.

the biosensor device IVB. with the substrate of being combined with pilin

The biosensor analysis device 32 that Figure 18 A and 18B explanation build according to one embodiment of the invention.The electronic property of the change that this devices use is observed by the pilin-peptide coating on metal detection plate 34.That is, plate 34 for example, is formed by the metal (stainless steel, tin, iron or titanium) when being coated with pilin peptide with the electron surface character of change.As above, by making plate surface be exposed to above pilin peptide 36 and the conjugate of analyte-bound fraction 38, or by compound is connected with the unconjugated pilin peptide of being combined, form pilin coating.Plate itself forms the lower surface of shallow biosensor reaction vessel 41, and the wall 40 of the sidepiece of described container in this device forms, and is partially filled waterborne conductive medium 42 in container.The pilin peptide that is attached to biosensor surface can comprise PEG-pilin peptide conjugate (PEG-D-pilin for example, its effect is the non-specific binding that reduces specimen material and plate) and the combination of the second peptide conjugate (RI-compound conjugate for example, its effect is to make the described compound from the teeth outwards covalently bound).

Biosensor circuit in this device comprises electrode 44, voltage source 46 and the reometer 48 stretching in container, wherein, contrary circuit connect be positioned at check-out console below.

Two kinds of surface configurations of Figure 19 and 20 explanation biosensors.In Figure 19, acceptor (part-combination) molecule (R) is covalently attached to pilin peptide (hook portion), pilin peptide then with biosensor surface covalent coupling.By the binding interactions of (i) pilin peptide and substrate surface, and (ii) impact of acceptor R on electroconductibility, determine the electric conductivity through biosensor substrate.For example, when ligand L (analyte) and receptors bind, lip-deep electronic property is further regulated, and causes the change of observed biosensor electric current.It is from larger electric current, to become less electric current after part and receptors bind that electric current shown in Figure 21 changes.

In Figure 20, by the covalent attachment of pilin/K spiral conjugate and substrate surface, interact with the coiled coil of acceptor-E spiral conjugate subsequently, acceptor R indirectly with substrate surface coupling.Three-component conjugate of the E spiral that the conjugate that in this embodiment, forms biosensor surface coating is pilin peptide and K spiral, put together with acceptor R and the ligand L that can be combined with R.In this embodiment, biosensor first with analyte response, analyte is combined, the K spiral site of masked surface with coating.This reaction can be used as the electric current change causing because of sheltering of K-spiral and is directly read, or can add E-spiral reagent to be combined with E-spiral in this stage, and after described E spiral and analyte response, still the amount of unshielded K-spiral is proportional.

Figure 22 A and 22B are the interactional voltmeter cyclic curve of biosensor (voltameter cycle curve), and wherein, His part (acceptor) is combined with substrate surface by the coiled coil configuration shown in Figure 20.Figure 22 A shows the complete cyclic voltammetry curve (cyclic voltalmetry curve) of the His part of showing on the K-spiral compound with E-spiral, and described E-spiral is covalently bound by pilin construct itself and stainless steel after adding specificity for the anti-His antibody of His part.Figure 22 B shows the enlarged view of the left-hand part of cyclic voltammetry curve, prove not only under positive bias but also in negative bias by antibody and His Binding change partly electric current.The combination of anti-His antibody and His acceptor produces minimum current cycle, proves that electric current declines with " analyte " and the combination of biosensor surface.When His acceptor obtains similar result by pilin peptide is direct when biosensor surface is combined, the analyte adopting is anti-His antibody.

In order to understand the running of sensor, consideration is useful from the corrosion resistance data of following table 2, table 2 shows Icorr, Ecorr, erosion rate and the Rp data of the following: (i) uncoated stainless steel plate (unmodified), (ii) use the stainless steel plate of pilin peptide (E-PAK) coating of puting together with E-spiral (electronegative leucine zipper) peptide, (iii) with the 3rd stainless steel plate (K-E-PAK) being coated with the identical conjugate (that is, peptide and E-spiral/K-spiral heterodimer are puted together) of the K-helical peptides combination of oppositely charged.Consider Icorr hurdle, this data presentation E-PAK closes its Icorr value of remarkable increase with hardening, with the viewed effect of PAK pilin contrary (in Table 9A) to independent.When conjugate is neutralized (K-E-PAK), Icorr value is significantly less than uncoated metal, is similar to the effect of observing for unconjugated peptide.From this data observation to the similar effect to Ecorr, erosion rate and Rp value, that is, and by significantly changing E-PAK pilin in conjunction with the surface action producing with the effect of E-spiral in the combination of the K spiral with oppositely charged.

Table 2. pilin conjugate is to stainless corrosion resistance data

From above explanation, can understand the various advantages of biosensor.First, because make the direct covalently bound pilin peptide of analyte bind receptor and biosensor surface affect the electronically active of biosensor surface, so the size and the charge generation that change surface complex by ligand binding directly act on, for example electric current reduces.The second, the interaction of molecule and metallic surface is fundamentally different from the interaction with plastics, because electronically active or the ability of metallic surface electron and molecule direct interaction determines interactional degree and power.The metal (for example gold) that does not form oxide skin has very active surface electronic (due to the fringing effect of crystal) the easy surface to them by absorbed.These materials are easy to from sample substrate non-specific adsorption protein and other molecules and therefore can not be used as biosensor platform.Metal (for example stainless steel) experience surface oxidation, to form the oxide skin (passivation) of passivation, at utmost reduces non-specific binding event and is not easy to make material and their surface bonding (so they can be widely used in medicine and food service industry).In the application of biosensor, use T4P17 peptide easily to make the ability that specific peptide/protein component is combined with the stainless steel of passivation give clear superiority to the signal to noise ratio of improving in detector ligand-acceptor interaction.As mentioned above, T4P17 is combined mediation transfer transport can play the effect of biosensor when being exposed to bias voltage with stainless steel.More than studies have shown that response part and the combination that is incorporated into pilin-acceptor conjugate of metallic surface of report regulate by the ability of the stream of electrons of biosensor surface.

RI type peptide and D type peptide provide competitive edge in biosensor application.Because RI peptide produces stronger electronic effect to EWF, show stronger delocalization effect, so when analyte or analyte-associated molecule and while being anchored to the receptors bind of substrate by pilin peptide, RI peptide can provide the electronic response of enhancing.On the other hand, D type peptide may provide more stable acceptor to connect, and applies by basad the specimen material that medium current allows to remove non-specific binding, as mentioned above.As mentioned above, sensor surface can be coated with the combination of two kinds of peptides (one of them or both have peg moiety) simultaneously.

the general analysis device IVE. with the substrate of being combined with pilin

Figure 23 A and 23B display analysis thing-detection means 16, it has plate 18, and the upper surface of detecting of plate 18 is coated with the conjugate of pilin peptide 20 and analyte-specific target molecule 22.Although this plate can be formed by the various materials of being combined with pilin peptide.But preferably for example stainless steel, tin, iron and titanium are surperficial with the covalently bound metal of pilin for they.This covalentlying bind in is easy to production, protein stability and coating stability aspect multiple advantage is provided.In described device, also comprise light beam source 24 (for example UV source) and photodetector 26 for detection surface (shown in 25).Peptide conjugate also can comprise peg moiety extraly, and it can play the effect that reduces surperficial non-specific binding.

In operation, the surface coverage of check-out console contains the fluid sample of target analytes, is shown as the analyte molecule 30 of solubility in Figure 12 B, and these are reacted with the lip-deep analyte-specific molecular 22 of detection.In the fluorimetric detector showing, reaction mixture can contain fluorescently-labeled antibody or can be specifically be incorporated into other binding reagents that detect surperficial analyte response.Then, washing detects surface to remove unconjugated component.The existence of the fluorescence of the combination on analytical reaction surface and level (fluorescence that shows as shown in Figure 12B, transmitting at 27 places) complete analysis now.

Be appreciated that fluorometric analysis easily can be incorporated in this device.For example, pilin peptide can contain fluorescence part and analyte-bound fraction can comprise that cancellation is effectively from the fluorescence quenching of the fluorescence of pilin fluorescence part.In this embodiment, the effect that quencher is sheltered in the combination of analyte and analyte bound fraction effectively produces stronger fluorescence when the combination of analyte and analyte bound fraction exists.

In an alternate embodiment, pilin peptide and analyte bound fraction can comprise respectively the first and second fluorescence classes, and when exciting under given excitation wavelength, they produce FRET (fluorescence resonance energy transfer) effectively.In this configuration, the combination of analyte and analyte binding substances class suppresses this type of energy effectively to be shifted, and reduces viewed fluorescence.

As mentioned above, D type peptide provides two clear superiorities in this application: with the more stable combination of substrate with pass through the ability that applied electric current is removed non-specific binding material.

the medical device V. with the metallic surface of coating

For example, studies have shown that about peptide and (stainless steel, tin, iron and the titanium surface) combination of some metallic surface of more than reporting, the Binding change metallic surface electronic property of pilin peptide, shows the formation of covalency between peptide and metal (electronics is shared) key.This discovery provides and has made bioactive molecules (for example peptide, lipid, nucleic acid, metabolite or drug molecule) be covalently attached to the new method on stainless steel, tin, iron and titanium surface, and has by pilin peptide and make bioactive compounds be covalently attached to the new medical device on the device metal surface of exposure.

Other metals of considering in described method are that periodic table of elements 4-6 is capable, the transition metal of 9-12 row, comprise cobalt, nickel, copper, zinc, ruthenium, rhodium, palladium, silver, cadmium, osmium, platinum, gold and mercury, their mixture and alloy, metalloid silicon and germanium and their oxide compound.

In the method, metallic surface is contacted with synthetic pilin peptide or PEG-pilin peptide conjugate, and described synthetic pilin peptide or PEG-pilin peptide conjugate contain derived from two sulphur rings of the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin and contain and be positioned at the N-end side of described ring or the 0-10 of C-end side or this two end side, 0-5 extra residue preferably.

If prepared pilin peptide or conjugate in advance to comprise covalently bound bioactive molecules, independent contact procedure causes bioactive molecules to be covalently attached to metallic surface.Preparing peptide (being pilin peptide in this example) is well-known with the method for the conjugate of bioactive molecules, comprise the fusion rotein that forms peptide and peptide biological activity molecule, and use specific chemical modification reaction to make bioactive molecules and the covalently bound reaction site of peptide to provide.For example, the final step in the solid-phase synthesis of pilin peptide can comprise and adds the reactive group (for example aldehyde) can be used for bioactive molecules covalent reaction.

Or, can after peptide is connected in metallic surface, make bioactive molecules react with pilin peptide or PEG conjugate, again adopt conventional bifunctional reagent or specific chemical group reactive chemistry so as to make bioactive molecules and metallic surface on the pilin covalent coupling of combination.

Another application (for generation of and purification of target polypeptides) by the first synthetic fusion rotein (for example, by recombinant polypeptide synthesis method) that contains the above-mentioned type pilin peptide and target polypeptides, implement.Then, this fusion rotein (its can by adding PEG group by further modification) is contacted with the solid carrier being formed by stainless steel, tin, iron, titanium, chromium, plastics, glass, silicate, pottery or their mixture, by pilin peptide moiety and being connected of carrier, fusion rotein is connected on carrier thus.

In wash vehicle, with after removing unconjugated material, use and can from the pilin peptide of combination, cut specifically the agent treated carrier of target polypeptides.This can comprise with the proteolytic ferment of determining the joint of sequence in cleavage of fusion proteins specifically processes carrier, or with the chemical or radiation energy processing carrier of the joint in can specificity cleavage of fusion proteins.Then, the target polypeptides discharging with the form of basic purifying wash-out or washing from carrier.

The Another application of combined techniques is to prepare the implantable device that has the surface properties of phase need or carry in its surface the bioactive molecules of phase need.For example, bone implant according to the present invention comprises stainless steel or titanium implant infrastructure, and a part for this structure is suitable for being positioned in bone district or facing to bone district.In order to promote being connected of bone and implant, for example, with the synthesis bacterium hairless protein peptide of PEG conjugate and the conjugate of bone morphogenic factor (RGD or bone morphogenic factor BMP2-BMP7) of the above-mentioned type, be coated with this part.

In relevant application, pilin peptide or PEG conjugate are put on to the surface of metal or polymer support, generation has the support of the present invention of the surface properties (tendency of for example less promotion surface reaction) of improvement, blood coagulation or scarring that the non-phase that described surface reaction can cause blood vessel to be implanted into site needs.Coating is alternately formed by the conjugate of pilin peptide or PEG-pilin peptide conjugate and bioactive molecules, for example, have pilin-Iimus drug conjugate that biology can releasing-joint (for example ester joint between pilin and medicine).

As what it will be appreciated that below, health has significantly been reduced in the metallic surface of coating can reach for device the Inflammatory response of (amount).

the medical device VI. with the biofilm formation of minimizing and the Inflammatory response of minimizing

Except the inflammation by infecting or cell injury/stress mediating, a large amount of iatrogenic inflammation are caused by medicine equipment.Tissue, cell and protein are exposed to non-biocompatible medical device and cause dysfunctional host response, and its clinical effect is underestimated greatly.Example comprises tissue reaction and the dysfunctional wound healing after medical prosthese (comprising blood vessel graft (vascular graft), artificial joint and other implantable device) inserts.Similarly, the activation of white corpuscle and blood coagulation system causes being regularly exposed to the critically ill patient's of extracorporeal circulation (for example cardiopulmonary bypass and hemodialysis) high incidence.These events further affect healing, regeneration and the rehabilitation of acute and patients with chronic diseases.

According to another aspect of the present invention, found the Inflammatory response of some metal (for example titanium) to using in medical device, by using the amino acids formed pilin peptide of the D-coating metal surfaces by the amino acid whose mixture of D-amino acid, D-and L-and contrary-inversion (RI) type, wherein all peptide forms can further be puted together with PEG.

In a research, make human peripheral blood mononuclear cell (PBMC) under the cell culture condition of standard separately or under the existence of titanium or steel plate (it is uncoated or be coated with D-type pilin peptide) incubation.In 37 ^oc incubation in RPMI substratum, after 24 hours, is analyzed the cytokine IL-1 β of substratum, and it is the indicator of Inflammatory response in PBMC.Analysis is as the tubulin of house keeper's contrast.Figure 25 A shows the IL-1 β of each mensuration and the western blotting of tubulin in five samples.As see, D-type pilin peptide coating suppresses the Inflammatory response in stainless steel and titanium sample effectively.In the column diagram shown in Figure 25 B, can quantitatively see this effect for titanium sample.

Carry out similarly studying the Inflammatory response to same sample with test person THP-1 scavenger cell, but comprise the extra sample being formed by stainless steel or titanium, it is coated with the control peptide 1 or 2 that represents uncombined pilin peptide sequence.Subsequently, in 37 ^oc makes coating and uncoated sample and THP-1 scavenger cell incubation 72 hours in RPMI substratum.Subsequently, analyze cytokine IL-1 β and the house keeping protein tubulin of substratum and cell lysate.Two kinds of western blottings in Figure 26 A have provided result.As see, independent titanium excites strong Inflammatory response, and it is significantly reduced by D-type pilin coating.The higher figure demonstration of the quantization degree for stainless steel sample providing in Figure 26 B, although quite little to uncoated stainless Inflammatory response,, D-peptide coating has suppressed this effect.From two control peptide samples (its show medium with strong reaction), can obviously find out, the effect of D-type pilin coating is quite specific.

On the one hand, the present invention includes the surperficial medical device that is exposed to Inflammatory response cell while having in implanting health, wherein, synthetic pilin peptide coating for these surfaces, described synthetic pilin peptide contains (i) derived from two sulphur rings of the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin, (ii) be positioned at the N-end side of described ring or the 0-10 of C-end side, 0-5 extra residue preferably, and (iii) by the D-amino acid of the amino acid whose mixture of D-amino acid, D-and L-or contrary-inversion (RI) type, formed.Described peptide can optionally be puted together with peg moiety.

Binding in Figure 24 A-24D has proved the strong combination of D-type pilin peptide and various medical device and device surface (comprising stainless steel (Figure 24 A), chromium cobalt support (Figure 24 B), latex catheter (Figure 24 C) and silicone ductus venosus (Figure 24 D)).In each example, sample is uncoated (the blank post in Figure 24), be coated with and contrast out of order pilin sequence (shade post) or D-type pilin peptide.Subsequently, the protein of washing sample the combination by above-mentioned HRT analytical method analytic sample.To all samples, in conjunction with the level of pilin peptide be significantly higher than the level of contrast, stainless steel shows and forms the highest coating of combination degree.The present invention includes other medical device of implanting or temporarily implanting experimenter's health, comprise metal and nonmetallic support, the conduit being formed by various flexible materials, at utensil and heart valve and other blood vessel valve of catheter tip supporting, wherein, described device comprises the surface of the Inflammatory response that is exposed in body and has been coated with D-type or RI type pilin peptide, and described peptide is optionally puted together with PEG.

At a related aspect, the present invention also comprises by suppressing implanting the method for the Inflammatory response of the medical device in experimenter with the exposed surface of synthetic pilin peptide coating device before implant devices, described synthetic pilin peptide contains (i) derived from two sulphur rings of the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin, (ii) be positioned at the N-end side of described ring or the 0-10 of C-end side or this two end side, the extra residue of 0-5 preferably, and (iii) by D-amino acid, the D-amino acid of the amino acid whose mixture of D-and L-or contrary-inversion (RI) type forms, wherein said pilin peptide can be optionally and peg moiety put together.

Again on the one hand, the present invention includes by the synthetic pilin peptide of D type or contrary-inversion is applied to the method that product surface stops or suppress the biofilm formation of non-pseudomonas bacterium on this product surface, the two sulphur rings that described synthetic pilin peptide contains derived from the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin are positioned at the N-end side of described ring or the 0-10 of C-end side or this two end side extra residue with containing, optionally put together the peg moiety on it, the amount of described peptide suppresses the biofilm formation of the non-pseudomonas bacterium of this class effectively.Preferred pilin peptide is D type peptide, and preferred peptide sequence contains the sequence that is defined as SEQ ID NO:10, for example, be defined as SEQ ID NO:3,4 or 9 sequence is illustrated.

Described goods can be for example non-medical device (dialysis membrane of metallic conduit or polymerization for example, it is experiencing biofilm formation between the normal usage period) or medical device (for example bone prosthese or the bone implant of the conduit of polymerization, metal or polymer support, heart valve or metal or pottery).More generally, the surface of the coating in goods can be the surface of metal (for example stainless steel, tin, iron or titanium), polymkeric substance, pottery or silicate.

In concrete example, described method can be used for suppressing Listera or staphylococcic biofilm formation, and the amount that is wherein applied to the pilin peptide of product surface suppresses respectively Listera or staphylococcic biofilm formation effectively.

In the research of following report, checked that D type or RI T4P pilin peptide block the ability that non-pseudomonas bacterium is combined with the bacterium of the stainless steel plate of coating.Experimentally, make stainless steel plate (uncoated contrast or with D type, RI or D type+RI T4P pilin peptide (K-122-4) coating), with 1 X 10 ⁵the bacterial cultures of CFU/ml bacterium in 10 mM PBS pH 7.2 be room temperature incubation 1 hour, jolting simultaneously.Then 10 mM PBS pH 7.2 washing 5 times for each plate, 5 ml/ washings, at room temperature with the moisture acridine orange dyeing of 1mM 2 minutes, wash with water and air-dry subsequently.Then by epifluorescence microscope art (epifluorescence microscopy), check each plate, and photographed in the 600 40X visuals field, measure the number of bacteria of the combination in each visual field.These conditions check the initial period of biofilm formation, and it is the prediction of setting up ripe biomembranous ability.

Figure 35 A shows that harmless Listera is attached to the result on plate, and proves that D type and RI pilin peptide all suppress the combination of the plate of harmless Listera and coating effectively.For monocyte Listeria monocytogenes strain H8,19 and J10 (being respectively Figure 35 B, 35C and 35D), D type peptide provides the remarkable protection for the combination of all 3 kinds of bacterial strains, and RI type only provides appropriate inhibition keying action.

With multiple staphylococcus, obtain similar results, shown in the data in Figure 36 A-36F, wherein D type peptide common (but not always) is to have more inhibition in two kinds of pilin peptides.

With Listeria monocytogenes and streptococcus aureus ( staphylococcus aureus) early research could not determine any difference in being combined with the bacterium of uncoated plate or the plate that is coated with L-type pilin peptide, although known L-type pilin peptide is blocked the combination of the plate of pseudomonas bacterium and coating effectively.In a word, result shows, although L-type T4P pilin peptide suppresses the biofilm formation of pseudomonas effectively, when coating surface, by D type and or RI T4P pilin peptide also can realize the inhibition to the biofilm formation of non-pseudomonas bacterium, wherein D type peptide has more inhibition conventionally.

Figure 37 A-37C shows the effect of PEG-D-pilin peptide to the biofilm formation on stainless steel surface.In each frame, the region of coating PEG-D-peptide demonstrates seldom or there is no biofilm formation, by contrast, in the uncoated region of metallic surface, has biofilm development clearly.Suppress to be applicable to pseudomonas aeruginosa strains (Figure 37 A) and streptococcus aureus.The research of carrying out supporting the present invention shows the restraining effect of PEG-D-pilin peptide coating to various other bacterial pathogens.These observationss are consistent with the bibliographical information of having delivered, described bibliographical information shows by adding the modification of PEG effects on surface effectively to limit biofilm formation, but to the business-like limitation of described method, is wherein that stable and long-acting PEG surface-treated builds.The present invention has solved this difficult problem by PEG is covalently attached to metallic surface via pilin peptide, and the verified described pilin peptide of the inventor itself can suppress to be coated with the lip-deep biofilm formation of peptide.

vII. the pipeline and the method that are coated with

The PEG conjugate of D-and RI type pilin peptide reduces frictional drag in the surface of coating and increases hardness and reduce the ability of corroding, in drilling well and pipeline industry (drilling and pipe industry), there is important application, for reducing the frictional drag that fluid flows through pipeline, increase hardness and corrosion resistance simultaneously.

In oil-gas exploration, conventionally liquid is pumped into basement rock structure under pressure, so that split and discharge buried Sweet natural gas in rock stratum, this process is called pressure break (fracking).Fracturing liquid (fracking fluid) is the slurries of water, propping agent (for example sand) and chemical additive normally.By with PEG-D or RI type pilin peptide conjugate pipe coating internal surface, the frictional drag that pumps into the slurries of this pipeline can be significantly reduced, and reduces costs and improves pump operated efficiency.Equally, for long distance, transport the pipeline of oil or gas product, with the pipeline with coating on inner surface, realize the remarkable improvement of carrying cost and efficiency.

Implement to process pipeline to reduce in the method for frictional drag of the fluid that flows through this pipeline, conjugate is introduced to this pipeline, described conjugate is following conjugate: (i) contain derived from two sulphur rings of the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin and contain and be positioned at the N-end side of described ring or the 0-10 of C-end side or this two end side extra D type of residue or the synthetic pilin peptide of contrary-inversion and (ii) polyalkylene glycol moiety, the amount of described conjugate is effectively by making described pilin peptide be connected in the frictional drag that duct wall reduces the fluid that flows through pipeline.Can in the independent coating solution of conjugate or suspension, conjugate be introduced to pipeline, or can for example, by peptide conjugate regularly being joined in the fluid (pressure break slurries) conventionally transporting by pipeline conjugate be introduced to pipeline.For example, conjugate regularly can be joined in pressure break slurries to whole solution be 1-10 gram/kilolitre, for initial coating with regularly recover coating.Be appreciated that the sand particle in slurries should use the precoating of PEG-pilin conjugate, as described below, to prevent that particle is attached to, be mostly intended to the conjugate adding for pipe coating internal surface and remove this most conjugate.

Preferred pilin peptide is the D type pilin peptide with the polyalkylene glycol moiety that is covalently attached to the C-end of described peptide or the one or both ends of N-end, and the molecular weight of the polyalkylene glycol moiety in wherein said peptide conjugate is between 0.2-500 kDal.

The inner surface of pipeline that pilin peptide conjugate connects can be metal, polymkeric substance, pottery or silicate surfaces.Exemplary metal comprises stainless steel, carbon steel, titanium, copper, brass, tin, iron, silver and magnesium and alloy thereof.When pipeline has stainless steel internal surface, by introducing the amount of the pilin peptide conjugate that is connected in inner surface of pipeline, be enough to reduce the corrosion of described internal surface.

Related fields of the present invention are the metals that have with one or more exposures of conjugate coating, polymkeric substance, the surperficial goods of pottery or silicate, described conjugate is following conjugate: (i) contain derived from two sulphur rings of the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin and contain and be positioned at the N-end side of described ring or the 0-10 of C-end side or this two end side extra D type of residue or the synthetic pilin peptide of contrary-inversion and (ii) polyalkylene glycol moiety, the amount of described conjugate is enough to reduce the frictional coefficient of one or more exposed surfaces.The goods of coating have the frictional coefficient of reduction, and the in the situation that of metal products, have larger corrosion resistance.Exemplary pilin peptide is D type peptide for example, and peg moiety as mentioned above.

One or more surfaces by exposing goods to be to be coated with solution or the suspension of PEG-pilin conjugate, to manufacture described goods, as above for as described in nonglycosylated pilin peptide coating.

When goods are while being its inner wall surface with the pipeline of peptide conjugate coating, the peptide conjugate of coating surface exists to be enough to reduce the amount of the frictional drag of the fluid that flows through pipeline.

When goods are that width dimensions is while being 100 microns or less microfluidic channel, vias inner walls surface is coated with peptide conjugate, the amount of described peptide conjugate is enough to reduce the frictional drag that fluid flows through passage, for example, and the frictional drag of the mortar by drilling rod (drilling pipe).

In another embodiment, the movable part of machine (for example gear (interdigitating gear) or the reciprocating element of mutual interlock) is coated with to reduce frictional drag and the wearing and tearing to parts with peptide conjugate.

In a further embodiment, described goods have surface, for example metallic lead or electrode, and it is applied to starting between assembling or locomorphic stage that protective lead avoids corrosion or to plumbous wearing and tearing (wear placed on the leads).

In another embodiment, the goods of coating are serous granules, sand for example, and it is coated with as follows: by PEG-D-or RI type pilin conjugate are joined in the suspension of described particle, or by making conjugate solution by grain bed, or simply by conjugate is joined in slurries.The frictional drag of the minimizing of particle, for particle-intergranular interaction with for the interaction between particle and inner-walls of duct, with respect to only reaching by pipe coating is only inner, has further reduced slurries by the resistance to flow of pipeline.

Although for specific embodiment and application, the present invention has been described,, should be appreciated that and can make various modifications not departing from situation of the present invention.

Claims

1. process pipeline to reduce the method for the frictional drag of the fluid that flows through pipeline, described method comprises:

To described pipeline, introduce the liquid that carries conjugate, described conjugate is following conjugate: (i) contain derived from two sulphur rings of the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin and contain and be positioned at the N-end side of described ring or the 0-10 of C-end side or this two end side extra D type of residue or the synthetic pilin peptide of contrary-inversion and (ii) polyalkylene glycol moiety, the amount of described conjugate is effectively by making described pilin peptide be attached to the frictional drag that tube wall reduces the fluid that flows through described pipeline.

2. the process of claim 1 wherein that described pilin peptide conjugate is the D type pilin peptide with the polyalkylene glycol moiety that is covalently attached to the C-end of described peptide or the one or both ends of N-end.

3. claim 1 or 2 method, the molecular weight of the polyalkylene glycol moiety in wherein said peptide conjugate is between 0.2-500 kDal.

4. the method for claim 1-3, the pilin peptide of wherein introducing in the described conjugate of pipeline contains the sequence that is defined as SEQ ID NO:10.

5. the method for claim 4, wherein introduces pilin peptide in the described conjugate of pipeline and contains and be defined as SEQ ID NO:3,4 or 9 sequence.

6. the method for claim 1-5, the accompanying inner surface of pipeline of wherein said pilin peptide conjugate is selected from metal, polymkeric substance, pottery or silicate surfaces.

7. the method for claim 1-6, wherein apply pilin peptide inner surface of pipeline be the metallic surface that is selected from stainless steel, carbon steel, titanium, copper, brass, tin, iron, silver and magnesium and alloy thereof.

8. the method for claim 1-7, wherein said pipeline has stainless steel internal surface and by the amount that described introducing is attached to the pilin peptide conjugate of inner surface of pipeline, is enough to reduce the corrosion of described internal surface.

9. the method for claim 1-8, the amount that is wherein attached to the pilin peptide conjugate of inner-walls of duct by described introducing is enough to reduce introduces the ducted frictional drag containing particle loaded fluid.

10. the method for claim 1-9, wherein said introducing comprises in the normal operation period described peptide conjugate regularly being joined to be introduced in ducted fluid.

11. have the goods of metal, polymkeric substance, pottery or the silicate surfaces of the one or more exposures that are coated with conjugate, described conjugate is following conjugate: (i) contain derived from two sulphur rings of the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin and contain and be positioned at the N-end side of described ring or the 0-10 of C-end side or this two end side extra D type of residue or the synthetic pilin peptide of contrary-inversion and (ii) polyalkylene glycol moiety, the amount of described conjugate is enough to reduce the surperficial frictional coefficient of described one or more exposures.

The goods of 12. claims 11, wherein said pilin peptide conjugate is the D type pilin peptide with the polyalkylene glycol moiety that is covalently attached to the C-end of described peptide or the one or both ends of N-end.

13. claims 11 or 12 goods, the molecular weight of the polyalkylene glycol moiety in wherein said conjugate is between 0.2-500 kDal.

The goods of 14. claim 11-13, the pilin peptide in wherein said conjugate contains the sequence that is defined as SEQ ID NO:10.

The goods of 15. claims 14, wherein introduce pilin peptide in the described conjugate of pipeline and contain and be defined as SEQ ID NO:3,4 or 9 sequence.

The goods of 16. claim 11-15, the surface of one or more exposures of wherein said goods is metallic surfaces.

The goods of 17. claim 11-16, the surface of one or more exposures of wherein said goods is stainless steel surface, and the described peptide conjugate that is coated with described surface exists to be enough to reduce the amount of the erosion rate on described surface.

The goods of 18. claim 11-17, described goods are pipelines of described peptide conjugate coating for its inner wall surface, and the described peptide conjugate that is coated with described surface exists to be enough to reduce the amount of the frictional drag of the fluid that flows through described pipeline.

The goods of 19. claim 11-17, described goods are that width dimensions is 100 microns or less microfluidic channel, and its inner wall surface exists with the amount that is enough to reduce fluid and flows through the frictional drag of described passage with the coating of described peptide conjugate and the described peptide conjugate that is coated with described surface.

The goods of 20. claim 11-17, described goods comprise sand particle, the frictional drag of pipeline is flow through in enough described peptide conjugate coating for its outside surface to reduce the particulate slurry of described coating.

The goods of 21. claim 11-17, described goods are the machines with movable part, surface of its coating peptide is moving contact each other.

The goods of 22. claim 11-17, described goods are the electron devices with the metallic surface of the exposure being coated with described peptide conjugate.

The goods of 23. claim 11-17, described goods are the orthopedic implant devices with stainless steel or titanium surface, and the described peptide conjugate that is coated with described surface exists to be enough to suppress the amount of biofilm formation on the surface of coating.

24. 1 kinds of goods, described goods comprise:

(i) have the surperficial element of exposure, it can be used as the substrate of biofilm formation under common working conditions by non-pseudomonas bacterium, and

(ii) with the D type of described surface bonding or the synthetic pilin peptide of contrary-inversion, the two sulphur rings that described synthetic pilin peptide contains derived from the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin are positioned at the N-end side of described ring or the 0-10 of C-end side or this two end side extra residue with containing, optionally be covalently attached to the polyalkylene glycol moiety of described peptide, the amount of described peptide suppresses the biofilm formation of the non-pseudomonas bacterium of this class effectively.

The goods of 25. claims 24, described goods are intended to the medical device in implantation or resident body, and the described pilin peptide that is wherein attached to described product surface is the D type pilin peptide that is conjugated to polyalkylene glycol moiety.

The goods of 26. claim 24-25, the molecular weight of the polyalkylene glycol moiety in wherein said peptide conjugate is between 0.2-500 kDal.

The goods of 27. claim 24-26, wherein said pilin peptide contains the sequence that is defined as SEQ ID NO:10.

The goods of 28. claim 24-27, wherein said pilin peptide contains and is defined as SEQ ID NO:3,4 or 9 sequence.

The goods of 29. claim 24-28, the surface of the described goods of being combined with described synthetic pilin peptide is selected from metal, polymkeric substance, pottery or silicate surfaces.

The goods of 30. claim 24-29, the surface of the described goods of being wherein combined with described synthetic pilin peptide is the metallic surface that is selected from stainless steel, carbon steel, titanium, copper, brass, tin, iron, silver and magnesium and alloy thereof.

The goods of 31. claim 24-30, the surface of the described goods of being wherein combined with described synthetic pilin peptide is stainless steel surface, and described pilin peptide and described surperficial covalent attachment.

The goods of 32. claim 24-30, described goods are used for suppressing Listera or staphylococcic biofilm formation, and the amount of the pilin peptide that wherein applied suppresses respectively Listera or staphylococcic biofilm formation effectively.

33. have in the method for surperficial metallic substance in grain boundary district of exposure in order to reduce the improvement of the erosion rate of described material in processing, and described improvement comprises:

The Surface Edge battery limit (BL) of the exposure in described material and the synthetic pilin peptide of D type or contrary-inversion are contacted, the two sulphur rings that described synthetic pilin peptide contains derived from the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin are positioned at the N-end side of described ring or the 0-10 of C-end side or this two end side extra residue with containing, optionally be covalently attached to the polyalkylene glycol moiety of described peptide, the amount of described peptide suppresses the erosion rate of described material effectively.

The improvement of 34. claims 33, wherein said pilin peptide conjugate is the D type pilin peptide with the polyalkylene glycol moiety that is covalently attached to the C-end of described peptide or the one or both ends of N-end.

The improvement of 35. claim 33-34, is wherein connected in the molecular weight of polyalkylene glycol moiety of described peptide between 0.2-500 kDal.

The improvement of 36. claim 33-35, wherein said pilin peptide contains the sequence that is defined as SEQ ID NO:10.

The improvement of 37. claim 33-36, wherein introduces pilin peptide in the described conjugate of pipeline and contains and be defined as SEQ ID NO:3,4 or 9 sequence.

The improvement of 38. claim 33-37, wherein said contact effectively makes the electronics work content in the grain boundary district of exposure be changed to few 0.3eV EFW unit and makes the hardness increase at least 20% in the grain boundary district of exposure, and the nano impress that described hardness is impacted metallic surface generation by the tip by atomic force microscope with given force is measured.

The improvement of 39. claim 33-38, wherein said metal is selected from stainless steel, carbon steel, titanium, copper, brass, tin, iron, silver and magnesium and alloy thereof.

The improvement of 40. claim 33-39, wherein said metallic substance is stainless steel, and described contact procedure makes the erosion rate of coating surface reduce at least 30%, described erosion rate is measured by the corrosion current through described surface.

The improvement of 41. claim 33-40, wherein said metallic substance is porous or netted, and implements described contact procedure so that the definite internal surface of described pilin peptide and hole by described material or mesh is combined.

The improvement of 42. claim 33-40; wherein said metallic substance has the grain boundary district of exposure; with the combination that effectively makes described pilin peptide and described grain boundary regioselectivity of described contact procedure, the surface by place, priority protection Qi grain boundary district strengthens hardness and the corrosion resistance of metallic surface thus.

43. 1 kinds of inhibition are to being designed to implant the method for the Inflammatory response of the medical device in experimenter, and described method comprises:

Before implanting described device, with synthetic pilin peptide, be coated with the surface of the exposure of described device, described synthetic pilin peptide contains (i) derived from two sulphur rings of the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin, (ii) be positioned at the N-end side of described ring or the 0-10 of C-end side or these two end sides extra residue, (iii) the D-amino acid that comprises contrary-inversion (RI) type, and optionally there is the polyalkylene glycol moiety that is covalently attached to described peptide.

The method of 44. claims 43, wherein said pilin peptide is the D type pilin peptide with the polyalkylene glycol moiety that is covalently attached to the C-end of described peptide or the one or both ends of N-end.

The method of 45. claim 43-44, is wherein connected in the molecular weight of polyalkylene glycol moiety of described peptide between 0.2-500 kDal.

The method of 46. claim 43-45, wherein said pilin peptide contains the sequence that is defined as SEQ ID NO:10.

The method of 47. claim 43-46, wherein introduces pilin peptide in the described conjugate of pipeline and contains and be defined as SEQ ID NO:3,4 or 9 sequence.

48. 1 kinds of biosensor devices for detection of analyte, described device comprises:

(a) conducting metal substrate, it has the synthetic pilin peptide of D type (RI) that (i) biosensor surface (ii) is combined with described substrate surface, the two sulphur rings that described synthetic pilin peptide contains derived from the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin are positioned at the N-end side of described ring or the 0-10 of C-end side or this two end side extra residue with containing, optionally there is the polyalkylene glycol moiety that is covalently attached to described peptide, (iii) be covalently attached to directly or indirectly the acceptor of described pilin peptide, and

(b) for detection of the detector of the change in electrical properties through substrate surface of the combination of the acceptor of response analysis thing associated ligands and surface bonding.

The biosensor device of 49. claims 48, wherein said biosensor substrate is formed by stainless steel, and pilin peptide combined thereon is D type pilin peptide.

The biosensor device of 50. claim 48-49, wherein said acceptor is by coiled coil E/K spiral to being covalently attached to pilin peptide, and one of described spiral centering is covalently attached to pilin peptide, and another is connected on acceptor.

51. make compound be covalently attached to the surperficial method of one or more exposures of the substrate being formed by stainless steel, tin, iron or titanium, and described method comprises:

The surface pilin peptide synthetic with D type of one or more exposures of described substrate contacted, the two sulphur rings that described synthetic pilin peptide contains derived from the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin are positioned at the N-end side of described ring or the 0-10 of C-end side or this two end side extra residue with containing, optionally there is the polyalkylene glycol moiety that is covalently attached to described peptide, make thus described pilin peptide covalently be attached to the surface of one or more exposures, and

Before or after described contact and combination, make described compound be covalently attached to described pilin peptide.

The method of 52. claims 51, wherein said substrate surface has the grain boundary district of exposure, and described contact procedure makes described compound preferentially be positioned at the place, grain boundary district of exposure effectively.

The method of 53. claim 51-52, wherein said pilin peptide is the D type pilin peptide with the polyalkylene glycol moiety that is covalently attached to the C-end of described peptide or the one or both ends of N-end.

The method of 54. claim 51-53, is wherein connected in the molecular weight of polyalkylene glycol moiety of described peptide between 0.2-500 kDal.

The method of 55. claim 51-54, wherein said pilin peptide contains the sequence that is defined as SEQ ID NO:10.

The method of 56. claim 51-55, wherein said compound is selected from peptide, oligosaccharides, lipid, nucleic acid and organic molecule.

The method of 57. claim 51-56, wherein said material is porous or netted, and described contact makes described pilin peptide and hole by described material or the definite internal surface of mesh be combined effectively.

58. 1 kinds of substrates, its surface bonding has the synthetic pilin peptide of (i) D type, the two sulphur rings that described synthetic pilin peptide contains derived from the C-end receptor binding protein of IV type Pseudomonas aeruginosa (T4P) pilin are positioned at the N-end side of described ring or the 0-10 of C-end side or this two end side extra residue with containing, optionally there is the polyalkylene glycol moiety that is covalently attached to described peptide, (ii) compound, described compound is covalently attached to described pilin peptide directly or indirectly.

The substrate of 59. claims 58, described substrate is formed by the metal that is selected from stainless steel, carbon steel, titanium, copper, brass, tin, iron, silver and magnesium and alloy thereof, and described substrate is as biosensor electroresponse element, wherein said compound by covalent linkage, be directly connected in described pilin peptide or by K/E spiral/spiral to being indirectly connected in the analyte receptor molecule of described pilin peptide, one in wherein said spiral is covalently attached to described pilin peptide, and another spiral is covalently attached to described compound.

The substrate of 60. claim 58-59, wherein said pilin peptide is the D type pilin peptide with the polyalkylene glycol moiety that is covalently attached to the C-end of described peptide or the one or both ends of N-end.

The substrate of 61. claim 58-60, is wherein connected in the molecular weight of polyalkylene glycol moiety of described peptide between 0.2-500 kDal.

The substrate of 62. claim 58-61, wherein said pilin peptide contains the sequence that is defined as SEQ ID NO:10.

The substrate of 63. claim 58-62, wherein introduces pilin peptide in the described conjugate of pipeline and contains and be defined as SEQ ID NO:3,4 or 9 sequence.

The substrate of 64. claim 58-63, described substrate is formed by stainless steel, and described substrate can be combined in the electronics work content that its surface has change because of described pilin peptide.